Decarboxylated cannabis resins, uses thereof and methods of making same

ABSTRACT

The disclosure relates to decarboxylated cannabis resins and methods of making the decarboxylated cannabis resins by extraction and decarboxylation of cannabinoids from Cannabis species using microwaves and solvents. The disclosure also relates to use of the decarboxylated cannabis resins for making pharmaceutical products comprising same.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application claims priority under the Paris Convention to U.S.Provisional Patent Application Ser. No. 62/356,262, filed Jun. 29, 2016,which is incorporated herein by reference as if set forth in itsentirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to decarboxylated cannabis resins and useof the decarboxylated resins directly as medicines or natural healthproducts, or as raw materials for making cannabis-derived products,including pharmaceutical products, pharmaceutical formulations andnatural health products. The disclosure also relates to products andcompositions comprising the decarboxylated cannabis resins. Alsodisclosed are methods of making the decarboxylated cannabis resins usingmicrowave extraction and decarboxylation.

BACKGROUND OF THE DISCLOSURE

Cannabis is a genus of flowering plants in the family Cannabaceae. Threespecies may be recognized (Cannabis sativa, Cannabis indica and Cannabisruderalis). Cannabis spp. contains a highly complex mixture ofcompounds, up to 568 unique molecules identified to date (Pertwee, R. G.e., Handbook of cannabis. Oxford University Press: Oxford, 2014).Cannabinoids are the most noteworthy compounds found in Cannabis spp.Among these compounds, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabinol(CBN), and cannabinodiol (CBDL) are known to be psychoactive (Pertwee,R. G. e., Handbook of cannabis. Oxford University Press: Oxford, 2014).Other cannabinoids such as cannabidiol (CBD), which is anon-psychoactive compound, exert their physiological effects through avariety of receptors including adrenergic and cannabinoid receptors(Pertwee, R. G. e., Handbook of cannabis. Oxford University Press:Oxford, 2014). Cannabinoid receptors (CB1 and CB2) belong to G-proteincoupled receptors (GPCRs) (Cottone, E.; Pomatto, V.; Cerri, F.;Campantico, E.; Mackie, K.; Delpero, M.; Guastalla, A.; Dati, C.;Bovolin, P.; Franzoni, M. F. Cannabinoid receptors are widely expressedin goldfish: molecular cloning of a CB2-like receptor and evaluation ofCB1 and CB2 mRNA expression profiles in different organs. FishPhysiology and Biochemistry 2013, 39 (5), 1287-1296). Phytocannabinoidsproduced by Cannabis spp. plants as well as endocannabinoids produced inour body bind to these receptors. Patients consume medical cannabis forthe treatment of or seek relief from a variety of clinical conditionsincluding pain, anxiety, epileptic seizures, nausea, and appetitestimulation (Galal, A. M.; Slade, D.; Gul, W.; El-Alfy, A. T.; Ferreira,D.; Elsohly, M. A. Naturally occurring and related syntheticcannabinoids and their potential therapeutic applications. Recentpatents on CNS drug discovery 2009, 4 (2), 112 and Downer, E. J.;Campbell, V. A. Phytocannabinoids, CNS cells and development: A deadissue? Phytocannabinoids have neurotoxic properties. Drug and AlcoholReview 2010, 29 (1), 91-98). It is also known that cannabinoids andendocannabinoids affect the development and maturation of neural cellsin early life (Downer, E. J.; Campbell, V. A. Phytocannabinoids, CNScells and development: A dead issue? Phytocannabinoids have neurotoxicproperties. Drug and Alcohol Review 2010, 29 (1), 91-98).Cannabinoid-like compounds isolated from flax have also shownanti-inflammatory properties (Styrczewska, M.; Kulma, A.; Ratajczak, K.;Amarowicz, R.; Szopa, J. Cannabinoid-like anti-inflammatory compoundsfrom flax fiber. Cellular & Molecular Biology Letters 2012, 17 (3),479-499).

Most cannabinoids are concentrated in a viscous resin produced instructures of the cannabis plant known as glandular trichomes. At least113 different cannabinoids have been isolated from the cannabis plant,the most common being Δ⁹-tetrahydrocannabinolic acid (Δ⁹-THC-A) which isthe non-active precursor of the psychoactive Δ⁹-tetrahydrocannabinol(Δ⁹-THC).

Δ⁹-THC-A is found in variable quantities in fresh, undried cannabis, butis progressively decarboxylated to Δ⁹-THC with drying, and especiallyunder intense heating such as when cannabis is smoked or cooked intocannabis edibles.

Δ⁹-THC is not the only substance obtained from cannabis with therapeuticbenefits to people. For example, studies have shown that cannabidiol(CBD), another major constituent of some strains of cannabis, has beenfound to be anxiolytic and to have antipsychotic properties, and may beneuroprotective in humans.

FIG. 1A shows chemical structures of six major cannabinoids present inor which can be obtained from cannabis: Δ ⁹-THC, Δ⁹-THC-A, Δ⁸-THC,cannabinol (CBN), Δ²-CBD and cannabidiolic acid (Δ²-CBD-A). FIG. 1Bshows the cannabinolic acid synthase pathway of cannabigerolic acid(CBGA) to cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA)and cannabichromenic acid (CBCA), which can all be decarboxylated toactive cannabinoid forms. In some cases, the non-decarboxylatedcannabinoids may also have therapeutic properties.

Decarboxylation is a chemical reaction that releases carbon dioxide andgenerates neutral cannabinoids. In one example in cannabis, thenon-psychoactive Δ⁹-THC-A can be converted to psychoactive Δ⁹-THC bydecarboxylation.

There is a need to produce a cannabis resin comprising cannabinoids thatare decarboxylated. The cannabis resins or extracts known in the art maynot be consistently and fully decarboxylated and comprise significantproportions of THCA and CBDA. For example, see WO 2014/159688. Thecannabis extracts or resins of the art are for use, for example, invaporization, infusion into edible matrices, and inhalation viaelectronic inhalation devices. Accordingly, the extracts and resins ofthe art comprise predominantly THCA and/or CBDA, as opposed to theirdecarboxylated counterparts (THC and CBD respectively), and it is onlyafter heating the extracts and resins via vaporization or cooking inedible matrices that decarboxylation occurs (i.e. only after heating theextracts and resins that the cannabinoids are decarboxylated into theiractive forms). As the cannabis resins of the present disclosure comprisedecarboxylated therapeutically active cannabinoids, they may be useddirectly. For example, the cannabis resins of the present disclosure maybe used directly in therapy or used to develop various cannabisformulations for pharmaceutical products that do not need to be furtherheated (i.e. decarboxylated) into therapeutically active forms.

Many people may be reluctant to use cannabis (either as a medicine ornatural health product) if it has to be “smoked” or heated (i.e.“vaping”). This may be because there are negative perceptions to smokingand vaping, especially in the older generation. Many people may be moreaccepting of cannabis if it is in a form that is more conventional (i.e.tablet form, or other conventional forms of medicine). In order, toproduce conventional forms of cannabis for use as a medicine or naturalhealth product, the cannabinoids in the product must be decarboxylated.

There is also a need to develop an efficient method of extracting anddecarboxylating cannabinoids from cannabis.

To date the simplest mechanism to perform decarboxylation of cannabinoidacids is by heating. Other processes of decarboxylation require toxicsolvents. In these systems, a distillation process such as fractionaldistillation may be employed to separate the organic solvents from thecannabis extract after decarboxylation.

In addition to decarboxylation, extraction of cannabinoids from theplant material is required. Extraction is most often done prior todecarboxylation. Mechanisms of extraction of cannabinoids such asΔ⁹-THCA and CBDA from cannabis that are known include sonication, reflux(Soxhlet) extraction and supercritical fluid extraction (SFE). Forexample, U.S. Pat. No. 9,044,390 describes a fractional SFE process ofcontacting cannabis plant material with a supercritical fluid solventsystem at a pressure between about 750 psi and 25,000 psi, and at atemperature between about −15° C. and 200° C.

With the rise in the authorized use of cannabis for medical, therapeuticand recreational purposes, there is an increasing need for areproducible method for obtaining a consistent activecannabinoid-comprising resin with known efficacy and to optimizeproduction and recovery of active decarboxylated cannabinoids. Withincreasing demand and potential uses, there is also a need for a methodthat can be easily scaled-up for manufacture of commercialpharmaceutical preparations, natural health product preparations orrecreational use preparations.

SUMMARY OF THE DISCLOSURE

The present disclosure provides a decarboxylated cannabis resin. Thedecarboxylated cannabis resin comprises the cannabinoid profile of theplant from which the resin was extracted, but the cannabinoids aredecarboxylated. The cannabinoids are at least 90%-100% decarboxylated(i.e. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or anyfraction thereof, e.g. 93.3% decarboxylated). Unlike some of thecannabis resins of the prior art, the decarboxylated cannabis resin ofthe present disclosure can be used directly, for example, in therapy orcan be used as a raw material to develop various pharmaceuticalformulations that do not need to undergo heating for decarboxylationinto therapeutically active forms.

In addition to the above decarboxylated cannabinoids, other chemicalsfound in Cannabis spp. and soluble in the solvent used, are found in theresin. Such compounds may include, for example, terpenes, fatty acids,chlorophyll, flavonoids, and other compounds. Some of the compounds mayundergo chemical transformation due to the processes used for extractionand decarboxylation. In some embodiments, THC and/or CBD are the majorcomponents of the decarboxylated cannabis resin.

The present disclosure also provides an improved method of extractingand decarboxylating cannabinoids from cannabis plant material, themethod comprising decarboxylating cannabis plant material before, duringor after extraction using microwave technology.

In some embodiments the method comprises: chopping down or grinding downthe cannabis plant material into smaller pieces to form cannabis plantmaterial of a size and form suitable for extraction; extractingcannabinoids from the broken down cannabis plant material by contactingthe broken down cannabis plant material with a solvent in a mixture toextract cannabinoids from the cannabis plant material anddecarboxylating the cannabinoids in the mixture by subjecting themixture to microwaves to form decarboxylated cannabinoids.

In some embodiments, the extraction and decarboxylation steps are donein one step, by exposing the plant material to a suitable solvent, (e.g.pharmaceutically acceptable solvent, for instance a solvent selectedfrom the group of solvents consisting of: 80-100% ethanol (for example,80%, 85%, 90%, 95%, 97%, 98%, 100%, and any integer or fraction of theinteger thereof), ethylene glycol, isopropanol or a combination ofsimilar solvents, and subjecting the plant material in the solvent tomicrowaves sufficient to obtain the desired decarboxylated cannabinoidcomprising resin. If 100% decarboxylation is not required, the durationof the microwaving step can be adjusted to obtain the desired degree ofdecarboxylated cannabinoids in the resin. In some embodiments, the plantmaterial in the solvent is placed in a microwave. In some embodiments itis subjected to microwaves for a time and/or at a pressure and/or at atemperature sufficient to produce desired decarboxylated (biologicallyactive) cannabinoid comprising resin. In some embodiments, the choppingdown, extraction and decarboxylation are done in one step.

In some embodiments when the extraction and decarboxylation steps aredone in one step, the broken down cannabis plant material is suspended(for example, by stirring, shaking or agitation) in a solvent andsubjected to microwaves resulting in an extracted and decarboxylatedcannabis resin.

An embodiment of the disclosure is a decarboxylated cannabis resin,wherein the resin comprises decarboxylated cannabinoids from a Cannabisspp. plant, such that Δ9-tetrahydrocannabinolic acid (Δ9-THCA) andcannabidiolic acid (CBDA) cannabinoids in the plant are eachindependently 90% to 100% decarboxylated to yieldΔ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) respectively in theresin. The range of 90% to 100% decarboxylation includes any integer orfraction of an integer in this range, for example 90%, 88.4%, and 100%.The decarboxylated cannabis resin may further comprise cannabinol (CBN),other decarboxylated cannabinoids, or any compounds (for example,terpenes, chlorophyll), including for example, the compounds found onTable 32.

The decarboxylated cannabis resin may comprise less than 5% solvent byweight or by volume, or it may be is substantially free of solvent.

An embodiment of the disclosure is a pharmaceutical compositioncomprising (i) the decarboxylated cannabis resin of the disclosure and(ii) a suitable pharmaceutically acceptable carrier or excipient.

Another embodiment of the disclosure is a natural health productcomprising the decarboxylated cannabis resin of the disclosure.

Another embodiment of the disclosure is a method of extracting anddecarboxylating cannabinoids from plant material, the method comprising:(i) extracting cannabinoids from the Cannabis plant material bycontacting the Cannabis plant material with a solvent to extractcannabinoids from the Cannabis plant material, the extract of Cannabiscomprising the solvent and cannabinoids; and (ii) decarboxylating thecannabinoids in (a) the plant material and (b) the extract comprisingcannabinoids by subjecting the plant material and extract to microwavesat temperature of about 100-200° C. in a sealed container and for a timeperiod sufficient to form the corresponding decarboxylated cannabinoidsin the extract. The temperature may be any integer or fraction of aninteger in the range of 100-200° C., for example 103° C., 175.5° C., and200° C.

In some embodiments, the plant material is broken down to form cannabisplant material of a size and form suitable for extraction before theextracting step.

In some embodiments, the extracting and decarboxylating occurconcurrently.

In some embodiments, the cannabis plant material is suspended in thesolvent by stirring or agitation.

In some embodiments, the solvent is selected from the group of solventsconsisting of: 80-100% ethanol, ethylene glycol, isopropanol, and acombination of any of the above. The ratio of solvent to cannabis plantmaterial may be such that the Cannabis plant material is submerged inthe solvent in the sealed container.

In some embodiments, the time period is about 15-75 minutes depending onscale of the reaction.

In some embodiments, the temperature is about 130° C. to about 180° C.,or about 150° C. to about 170° C.

In some embodiments, the microwaves have a frequency of about 2.45 GHzand a wavelength of about 1.22×10⁸ nm.

In some embodiments, subjecting the plant material and extract tomicrowaves in the sealed container occurs under pressure of about 1-22bar. The pressure may be any integer or fraction of an integer in therange of 1-22 bar, for example 1 bar, 15.5 bar and 18 bar.

In some embodiments the extracted and decarboxylated cannabinoids arerecovered in the form of a cannabis resin or in the form of isolatedcompounds. For example, the decarboxylated cannabinoids may be recoveredfrom the cannabis resin by separating (i) a single cannabinoid, (ii) amixture of cannabinoids, (iii) a single compound or (iv) a mixture ofcompounds from the resin. The decarboxylated cannabinoids may berecovered using a Celite® and/or an activated charcoal carbon filter.

In some embodiments, the decarboxylated cannabinoids in the resin may besubjected to winterization for the removal of waxes.

In some embodiments, the cannabis plant material is dried prior to thebreaking down step. In some embodiments, the cannabis plant material isdried cannabis.

In some embodiments, a further step of removing the solvent from theresin to obtain a substantially solvent-free decarboxylated cannabisresin is used.

In some embodiments, the cannabis plant material is trichomes, cannabisfemale inflorescence, flower bract, cannabis stalk, cannabis leaves, orcombinations thereof.

In some embodiments, the solvent is removed from the resin.

An embodiment of the disclosure is the method of the disclosure done ona commercial scale.

An embodiment of the invention is the use of the cannabis resin of thedisclosure in a natural health product or in a pharmaceutical product.

Additional aspects of the present application will be apparent in viewof the description which follows. It should be understood, however, thatthe detailed description and the specific examples, while indicatingembodiments of the disclosure, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe disclosure will become apparent to those skilled in the art fromthis detailed description.

Abbreviations

Δ9-THC=Δ9-Tetrahydrocannabinol, CBD=Cannabidiol, CBN=Cannabinol,CBDL=Cannabinodiol, CBC=Cannabichromene, CBL=Cannabicyclol,CBE=Cannabielsoin, CBG=Cannabigerol, THV=Tetrahydrocannabivarin,CBDV=Cannabidivarin, CBCV=Cannabichromevarin,THCA=Tetrahydrocannabinolic acid, CBDA=Cannabidiolic acid,CBNA=Cannabinolic acid, CBCA=Cannabichromenic acid, CBLA=Cannabicyclolicacid, CBEA=Cannabielsoic acid, CBGA=Cannabigerolic acid,THCVA=Tetrahydrocannabivarinic acid, C1-THCRA=Tetrahydrocannabiorcolicacid, C4-THCA=C4-Tetrahydrocannabinolic acid, CBND=Cannabinodiol.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the subject matter may be readily understood, embodimentsare illustrated by way of examples in the accompanying drawings, inwhich:

FIG. 1A shows chemical structures of six major cannabinoids present incannabis: Δ ⁹-THC, Δ⁹-THC-A, Δ⁸-THC, CBN, Δ²-CBD and Δ²-CBD-A;

FIG. 1B shows the cannabinoic acid synthase pathway of cannabigerolicacid (CBGA) to cannabidiolic acid (CBDA), tetrahydrocannabinolic acid(THCA) and cannabichromenic acid (CBCA);

FIG. 2A shows medical cannabis and decarboxylated cannabis resin

FIG. 2B is a mass spectra of Strain 1 before decarboxylation of medicalcannabis

FIG. 2C is a mass spectra of Strain 1 after decarboxylation of medicalcannabis

FIG. 3 is a block diagram illustrating a process for extracting anddecarboxylating cannabinoids from cannabis plant material according toone embodiment of the application;

FIG. 4 shows a standard curve for Δ⁸-THC (selected ion recording (SIR)chromatograms were integrated and the AUC was plotted vs. concentration(ug/mL);

FIG. 5 shows a standard curve for Δ⁹-THC (selected ion recording (SIR)chromatograms were integrated and the AUC was plotted vs. concentration(ug/mL);

FIG. 6 shows a standard curve for THCA (selected ion recording (SIR)chromatograms were integrated and the AUC was plotted vs. concentration(ug/mL);

FIG. 7 shows a standard curve for CBD (selected ion recording (SIR)chromatograms were integrated and the AUC was plotted vs. concentration(ug/mL);

FIG. 8 shows a standard curve for CBDA (selected ion recording (SIR)chromatograms were integrated and the AUC was plotted vs. concentration(ug/mL);

FIG. 9 shows a standard curve for CBN (selected ion recording (SIR)chromatograms were integrated and the AUC was plotted vs. concentration(ug/mL);

FIG. 10 shows a relative amount of Δ⁹-THC and THC-A present in cannabisplant material samples extracted using microwave radiation at varioustemperatures;

FIG. 11 shows concentrations of various cannabinoids in the cannabisextracts using ethanol, Soxhlet and SFE, without subjecting cannabis tomicrowave conditions;

FIG. 12 shows concentrations of various cannabinoids in the cannabisextracts using ethanol, Soxhlet and SFE followed by microwave heating;

FIG. 13 shows various cannabinoid concentrations from cannabis aftermicrowave heating only at various temperatures;

FIG. 14 shows various cannabinoid concentrations from cannabis aftermicrowave heating and subsequent SFE extraction at various temperatures;

FIG. 15 shows various cannabinoid concentrations from cannabis aftermicrowave heating and subsequent SFE extraction at various temperatures,where the acidic and neutral form of THC are added together to representthe total THC extracted by each method;

FIG. 16 shows a chromatogram of mass scan in a positive mode (150-500m/z) of cannabis extract obtained from microwave extraction at 170° C.,15 min;

FIG. 17 shows single ion recording (SIR) (+ve) detection of m/z=315 forcannabis extract obtained from microwave extraction at 170° C., 15 min;

FIG. 18 shows SIR (+ve) detection of m/z=359 for cannabis extractobtained from microwave extraction at 170° C., 15 min;

FIG. 19 shows a chromatogram of a mass scan in a negative mode (150-500m/z) of cannabis extract obtained from microwave extraction at 170° C.,15 min;

FIG. 20 shows SIR (−ve) detection of m/z=313 for cannabis extractobtained from microwave extraction at 170° C., 15 min;

FIG. 21 shows SIR (−ve) detection of m/z=357 for cannabis extractobtained from microwave extraction at 170° C., 15 min;

FIG. 22 shows a chromatogram of mass scan in a positive mode (150-500m/z) of cannabis extract obtained from microwave extraction at 130° C.,10 min;

FIG. 23 shows SIR (+ve) detection of m/z=315 for cannabis extractobtained from microwave extraction at 130° C., 10 min;

FIG. 24 shows SIR (+ve) detection of m/z=359 for cannabis extractobtained from microwave extraction at 130° C., 10 min;

FIG. 25 shows a chromatogram of mass scan in a negative mode (150-500m/z) of cannabis extract obtained from microwave extraction at 130° C.,10 min;

FIG. 26 shows SIR (−ve) detection of m/z=313 for cannabis extractobtained from microwave extraction at 130° C., 10 min; and

FIG. 27 shows SIR (−ve) detection of m/z=357 for cannabis extractobtained from microwave extraction at 130° C., 10 min

FIG. 28A Overview of select signals as seen in the mass spectra beforedecarboxylation of cannabis extract

FIG. 28B Overview of select signals as seen in the mass spectra afterdecarboxylation of cannabis extract

FIG. 29A-D Shows flow charts of various extraction methods. FIG. 29AUltrasonication; FIG. 29B Sohxlet extraction; FIG. 29C SupercriticalFluid Extraction (Omar, J. Olivares, M. et al. J. Sep. Sci. 2013, 36,1397-1404, incorporated herein by reference); FIG. 29D Closed systemmicrowave extraction (in ethanol, hexane or isopropanol or liquid CO₂),as described in experiments 5-9 below.

DETAILED DESCRIPTION

As previously described, tetrahydrocannabinol (THC), or more preciselyits main isomer Δ⁹-THC, is the principal psychoactive constituent (orcannabinoid) of cannabis. Unlike THC, its corresponding acid THC-A (orΔ⁹-THC-A for its main isomer) is a non-psychoactive cannabinoid found inraw and live cannabis.

Δ⁹-THC is only one of the family of compounds known as cannabinoids. Forexample, Δ⁸-THC is a double bond isomer of Δ⁹-THC and is a minorconstituent of most varieties of cannabis. The major chemical differencebetween the two compounds is that Δ⁹-THC is easily oxidized tocannabinol (CBN) whereas Δ⁸-THC does not oxidize to cannabinol as it isvery stable. Δ⁸-THC, for the most part, produces similar psychometriceffects as does Δ⁹-THC, but is generally considered to be 50% lesspotent than Δ⁹-THC. On the other hand, CBD has no or limitedpsychometric activity on its own when administered to humans, however asdiscussed below CBD comprises other therapeutic qualities. In thecannabis plant, CBGA is a precursor and cannabinol (CBN), a metaboliteof THC. As cannabis matures, THC gradually breaks down to CBN which alsohas psychoactive properties.

FIG. 1 shows the chemical structures of six major cannabinoids presentin cannabis: Δ ⁹-THC, Δ⁹-THC-A, Δ⁸-THC, CBN, Δ²-cannabidiol (Δ²-CBD orCBD) and Δ²-cannabidiolic acid (Δ²-CBD-A or CBD-A).

FIGS. 2a, 2b and 2c show the decarbosylated cannabis resin and the massspectra of Strain 1 before (b) and after (c) decarboxylation of medicalcannabis, and indicates 100% conversion of CBDA and THCA into CBD andTHC, respectively.

Turning to FIG. 3, a method 100 for extracting and decarboxylatingcannabinoids from cannabis plant material is shown. Generally, method100 comprises drying raw cannabis plant material at step 102, breakingdown the cannabis plant material to form cannabis plant material of asize and form suitable for extraction at step 104, extractingcannabinoids from the broken down cannabis plant material by contactingthe broken down cannabis plant material with a solvent to extract thecannabinoids from the cannabis plant material and decarboxylating thecannabinoids by subjecting the extract to microwaves to formdecarboxylated cannabinoids. Decarboxylation of the cannabinoids mayoccur when the cannabinoids are in the plant, when the cannabinoids arein the extract, or both. In some embodiments the extract is subjected tomicrowaves in a closed reaction vessel at a temperature and timesufficient to form decarboxylated cannabinoid.

The extracted and decarboxylated cannabinoids are optionally recovered.Recovery can include filtering the solvent from the extract of cannabisplant material to isolate the decarboxylated cannabinoids.

In some embodiments, the process comprises an extraction step before,during or after decarboxylation. In some embodiments, the process of thedisclosure comprises more than one extraction step.

In some embodiments, the decarboxylation step can occur before, duringor after extraction.

In an embodiment of the disclosure, a one-step method for extraction anddecarboxylation is used, wherein the cannabis plant material (that issuitably prepared, e.g. optionally dried and broken down as describedherein) is placed in a suitable extracting solvent (e.g. apharmaceutically acceptable solvent such as ethanol, glycerol, andisopropanol, and other solvents as is known to those skilled in the art,kept in suspension or solution (e.g. by stirring, agitation, shaking orother means known to those skilled in the art) and subjected tomicrowave radiation while stirring at a temperature, pressure and timeto obtain suitably extracted and decarboxylated cannabinoids that can berecovered for use as either a mixture or individual chemical components(e.g. for use as a therapeutic or pharmaceutical product). Further insome embodiments, the solvent can be food grade oil and/or a mediumchain triglyceride, for example, coconut oil.

Such a method is more efficient in converting the cannabinoid acid intoits decarboxylated form than other known methods of extraction anddecarboxylation (such as simple heating).

Particularly, FIGS. 11 and 12 show various concentrations of differentcannabinoids in cannabis plant material extracts using ethanol, Soxhletand SFE, without subjecting cannabis to microwave conditions andfollowed by microwave heating, respectively. By comparing concentrationsmeasured through the use of solvent extraction alone, Soxhlet extractionalone (e.g. solvent and heat) and SFE alone (e.g. solvent and heat), asshown in FIG. 11, with concentrations measured by using solventextraction followed by microwave, Soxhlet extraction followed bymicrowave and SFE followed by microwave, as shown in FIG. 12, addingmicrowave heating to each of solvent extraction, Soxhlet extraction andSFE can greatly increase THC, CBD and CBN concentrations and decreaseTHCA concentration in the extract.

FIGS. 13 and 14 further show that high concentrations of THC, CBD, CBNand THCA in the extract are achieved when the extract is exposed tomicrowave radiation at about 170° C. for about 20 minutes. Furtherdetails regarding working variables are provided below.

Drying 102

At step 102, cannabis plant material can be dried to reducewater/moisture content. Herein, cannabis plant material encompasses anycannabis plant, including but not limited to Cannabis sativa, Cannabisindica and Cannabis ruderalis, and all subspecies thereof (for example,Cannabis sativa subspecies indica including the variants var. indica andvar. kafiristanica), including wild or domesticated type Cannabis plantsand also variants thereof, including cannabis chemovars (varietiescharacterized by virtue of chemical composition) which naturally containdifferent amounts of the individual cannabinoids and also plants whichare the result of genetic crosses, self-crosses or hybrids thereof. Theterm “cannabis plant material” is to be interpreted accordingly asencompassing plant material derived from one or more cannabis plants.“Cannabis plant material” includes live or fresh cannabis and driedcannabis. In addition, any part of the cannabis plant may be used,including but not limited to trichomes, flower buds, flower bracts,leaves, stalk and any other plant part that may contain cannabinoids.Also, although the female plants may produce a higher concentration ofcannabinoids than male plants, both female (including “feminizedplants”) and male plants can be used.

Optional drying step 102 can be used to remove excess moisture from thecannabis plant material prior to the cannabis plant material undergoingextraction and decarboxylation. Removing water content from the cannabisplant material can help to provide even heating at later stages in theextraction and/or decarboxylation process. Alternatively, fresh cannabisplant material (e.g. from the plant directly) can be used for thesubsequent break down, extraction and decarboxylation steps. Herein,drying of the cannabis plant material at step 102 can occur by anymeans, for example in an oven at temperatures in the range of 60-75 C,or similar conditions, or using a vacuum oven or similar conditions overseveral hours, for example 4 hours, or 6 hours or 8 hours, depending onthe amount of moisture. During the drying process, when heating isemployed using heating elements in the ovens or infrared heating amongother processes, some of the cannabinoid carboxyl acids forms could beconverted into their decarboxylated cannabinoid forms.

Break Down 104

At break down step 104, cannabis plant material can be broken down toproduce a cannabis plant material of a size and form suitable forextraction and decarboxylation by subjecting to microwave heating.

Trichomes (i.e. resin glands) of the cannabis plant material are nearlymicroscopic, mushroom-like protrusions from the surface of the buds, fanleaves, and the stalk. While relatively complex, trichomes are comprisedprimarily of a stalk and a head. The production of cannabinoids such asTHC occurs predominantly in the head of the trichome. Cannabinoids areconcentrated in the trichomes of the plant. The trichome is built toeasily shed from the cannabis plant material surface.

The term “a size and form suitable for extraction and decarboxylation”refers to a reduction in the particle size of the cannabis plantmaterial fragments.

Herein, breakdown of the cannabis plant material at step 104 can occurby any mechanical means including by crushing, smashing, grinding,pulverizing, macerating, disintegration or equivalent processes as areknown to those skilled in the art that reduce the cannabis plantmaterial into small pieces suitable in size and form for extractionand/or decarboxylation.

In one example embodiment, sonication can also be used to loosen thecannabis plant material in contact with an appropriate solvent such asethanol, and/or by breaking down cellular membranes making it suitablefor extraction and/or decarboxylation. In another example embodiment,maceration can be performed with a mortar and pestle to produce acannabis plant material of a size and form suitable for extractionand/or decarboxylation.

In certain embodiments, the cannabis plant material is reduced in sizesuch that its particle size is within a range of 1 mm to 10 mm.

Extraction 106

Upon completion of break down step 104, extraction step 106 may beperformed. It should be noted that extraction step 106 may occur as aseparate step to decarboxylation step 108 either before or afterdecarboxylation step 108, or as will be described below, extraction step106 and decarboxylation step 108 may occur concurrently. For theavoidance of doubt it should be understood that any extraction,including but not limited to sonication in the presence of a solvent,reflux (Soxhlet) extraction and supercritical fluid extraction (SFE) mayoccur before or after decarboxylation step 108. In addition, it shouldalso be understood that break down (104), extraction (106) anddecarboxylation (108) may also occur in one step.

In one embodiment, extraction step 106 can comprise contactingcannabinoids from the broken down cannabis material that is the productof break down step 104 with a solvent.

In some embodiments, the solvent treatment in extraction step 106removes non-cannabinoid impurities to leave a substantially purepreparation of cannabinoids. It has been shown that non-polar, liquidsolvents may be useful for this function. Suitable non-polar solventstherefore include essentially any non-polar solvents which aresubstantially less polar than the cannabinoids, such that impuritieswhich are more polar than the cannabinoids are removed by treatment withthe solvent. Filtration and other methods as is known to those skilledin the art can also be used to remove impurities.

Useful non-polar solvents include, but are not limited to, C5-C12straight chain or branched chain alkanes, or carbonate esters of C1-C12alcohols. The more volatile C5-C12 alkanes may be particularly useful,as they are more easily removed from the extract. Further, solvents thathave been approved for use in pharmaceutical compositions, such asethanol (e.g. 95% ethanol) may be particularly useful.

Particularly useful solvents include pentane, hexane, heptane,iso-octane and ethanol, and/or mixtures thereof or the like as is knownto those skilled in the art.

In one embodiment of extraction step 106, broken down cannabis plantmaterial can be added to a solvent and concurrently sonicated.

Herein, sonication refers to the application of ultrasonic vibration(e.g. >20 kHz) to fragment cells, macromolecules and membranes of thedried or undried cannabis plant material. Ultrasonic vibration can beprovided by any means known in the art.

In one exemplary embodiment, sonication of a mixture of cannabis plantmaterial and solvent can occur for 5-25 minutes at 25° C., where theratio of cannabis plant material and solvent is such that all cannabisplant material is submerged in the solvent completely in the reactionvessel.

Upon the completion of sonication of the mixture of cannabis plantmaterial and solvent, the solvent is removed from the mixture. Removalof the solvent can occur by any means known in the art, including butnot limited to filtration and/or evaporation. One embodiment forfiltering after sonication is vacuum filtering over a glass sinteredfunnel to separate the resultant extract in the filtrate and the plantmaterial. The latter can then be subjected to further extractions suchas Soxhlet or other solvent extractions as is known to those skilled inthe art, for example, SFE.

In yet another embodiment of extraction step 106, cannabinoids can beextracted from cannabis plant material that is broken down in step 104by reflux (Soxhlet) extraction.

During reflux (Soxhlet) extraction, cannabis plant material that isbroken down in step 104 is generally suspended above a heated solvent ina receptacle. The solvent is heated to reflux in a distillation flasksuch that solvent vapor travels up a distillation arm and floods intothe receptacle housing raw cannabis material. A condenser suspendedabove the raw cannabis material ensures that any solvent vapor risingabove the raw cannabis material cools and subsequently drips back downinto the receptacle housing the raw cannabis material. The receptacleslowly fills with warm solvent such that cannabinoids begin to dissolveinto the warm solvent. When the receptacle fills, it is emptied by asiphon such that the solvent is returned to the distillation flask. Thiscycle may be allowed to repeat many times, over hours or days.

Preferably, reflux (Soxhlet) extraction occurs at a solvent temperaturehigher than the boiling point of the corresponding solvent used forextraction and is conducted over a period of approximately 3 to 5 hours.

Once extraction is complete, removal of the solvent can occur by anymeans known in the art, including but not limited to filtering and/orevaporation as previously described.

In place of either sonication or reflux (Soxhlet) extraction asdescribed above, another embodiment of extraction step 106 encompassedby the subject application is the extraction of cannabinoids fromcannabis plant material by SFE.

SFE refers to a process of separating one or more components(extractant) from another (matrix) using supercritical fluids as theextracting solvent. Extraction is usually from a solid matrix (e.g.cannabis plant material), but can also be from liquids or resinousmaterial (for example, hash oil).

Although numerous supercritical fluids can be used, carbon dioxide (CO₂)is the most commonly used supercritical fluid for SFE. In otherexemplary embodiments, CO₂ can be modified by co-solvents such asethanol or methanol as is known to those skilled in the art.

Extraction conditions for supercritical fluids are above the criticaltemperature (for example, 31° C. for CO₂) and critical pressure (forexample, 74 bar for CO₂). Addition of modifiers such as but not limitedto ethanol can require altering these extraction conditions.

An exemplary SFE system contains a pump for CO₂ (as well as any othersolvents), a pressure cell to contain the cannabis material, a means ofmaintaining pressure in the system and a collecting vessel. The liquidis pumped to a heating zone, where it is heated to supercriticalconditions. It then passes into the extraction vessel, where it rapidlydiffuses into the solid matrix and dissolves the cannabis material to beextracted. The dissolved material (for example, cannabinoids) is sweptfrom the extraction cell into a separator at lower pressure, and theextracted material settles out. The CO₂ can then be cooled,re-compressed and recycled, or discharged to atmosphere.

Herein, the temperature of the SFE extraction performed at extractionstep 106 can, in some embodiments, be in the range of 35-55° C.

Further the pressure the SFE extraction performed at extraction step 106can in some embodiments be in the range of 65-85 bar.

SFE in the present disclosure occurs at about 40° C. with a backpressure regulator pressure of 12 MPa and the extracted compounds aremonitored using a photodiode array of 200-600 nm (monitoring at 254 nm).The acquisition time and method times of the system can each vary by afew minutes up to 60 minutes, ideally between 15 and 30 minutes,depending on the ratio of supercritical fluid and the co-solvent isaltered for the extraction.

In specific embodiments, SFE can be carried out multiple times insuccession. In such embodiments, the SFE is a fractional SFE.

As previously described for sonication with a solvent and reflux(Soxhlet) extraction, once SFE is complete, removal of the solvent canoccur by any means known in the art, including but not limited tofiltering and/or evaporation.

Microwave-Assisted Extraction and Decarboxylation 108

Decarboxylation of phytocannabinoid acids such as Δ⁹-THC-A is a functionof the time and temperature of the reaction. For instance, thedecarboxylation of concentrated Δ⁹-THC-A in solution into Δ⁹-THC and thedegradation of Δ⁹-THC vary with temperature. Temperature controls aretherefore important for controlling desired ratios of decarboxylationproducts. The use of conventional household microwaves in the processingof cannabis has been discussed in the literature, however, with mixed,inconsistent results and not necessarily specifically for extraction ina solvent and decarboxylation. Further, in order to obtain 100%decarboxylation, the temperature must be sustained over a period of timewithout burning of the cannabis material or boiling/evaporation of thesolvent. If the temperature is higher than the boiling point of thesolvent employed, the solvent will boil over and/or evaporate. In orderto sustain the temperature over the required period of time to fullydecarboxylate the cannabinoids but not burn the cannabis plant materialor boil/evaporate the solvent with the cannabis, the microwave vessel(i.e. the sealed container) must be under pressure. Sealing the vesselor container ensures pressure in the vessel or container.

As shown in FIG. 3, microwave-assisted extraction can occur eitherimmediately after mechanical breakdown step 104 or after a preliminaryextraction step 106 as described above.

Microwave assisted extraction and decarboxylation 108 can comprisesuspending cannabis plant material in a solvent and subjecting themixture to microwaves in a closed container at a temperature, pressureand time sufficient to form decarboxylated cannabinoids.

Herein, the term “microwaves” refer to a form of electromagneticradiation with wavelengths ranging from one meter to one millimeter;with frequencies between 300 MHz (100 cm) and 300 GHz (0.1 cm).

Further, solvent treatment in microwave assisted extraction anddecarboxylation step 108 is again to remove non-cannabinoid impuritiesto leave a substantially pure preparation of cannabinoids. As such,non-polar, liquid solvents are useful for this function. In oneembodiment, ethanol is used as the liquid solvent in microwave assistedextraction and decarboxylation step 108. In another embodiment, 95%ethanol is used as the liquid solvent in microwave assisted extractionand decarboxylation step 108.

As ethanol has a boiling point of 78° C. and the decarboxylation processof cannabis is temperature dependent (as described above), temperaturecontrol is important in microwave-assisted extraction step 108.

In one exemplary embodiment, suitable conditions to promotedecarboxylation of CBDA and THCA to CBD and THC, respectively, are tosuspend the cannabis plant material in a solvent (such as ethanol) andthen subject this mixture to electromagnetic radiation (for example,microwaves) of a wavelength in the range of 10⁶-10⁹ nm and a frequencyof 300 MHz-300 GHz. In one embodiment, the conditions further include,for example, the following: temperature range of 40-250° C., temperatureincrease of 2-5° C./sec, pressure range of 0-20 bar (2 MPa, 290 psi),microwave power range of 0-400 W at 2.45 GHz or minor variations andadjustments to suit a particular solvent and/or reaction conditions toreach the required temperature and accomplish decarboxylation. Ifstirring is required, a variable magnetic stirrer (300-900 RPM) may beused.

In another exemplary embodiment, microwave assisted extraction anddecarboxylation can be performed at a temperature in the range of100-200° C. (including all possible integers and fractions of integersin this range, for example, 163.5° C.), 130-170° C., 150-170° C. or130-150° C.

In another exemplary embodiment, microwave assisted extraction anddecarboxylation can be performed at a pressure in the range of 2-22 bar(including all possible integers and fractions of integers in thisrange), for example, 10.7 bar or 17 bar or 18 bar or 19 bar or 20 bar or21 bar.

The cannabis plant material can be suspended in a solvent and subjectedto microwaves at frequency and wavelength of 2.45 GHz and 1.22×10⁸ nm,respectively, and a temperature in the range of 130-190° C.

The raw cannabis material can be suspended in a solvent and subjected tomicrowaves at frequency and wavelength of 2.45 GHz and 1.22×10⁸ nm,respectively, and a temperature in the range of 150-190° C. and thesolvent is ethanol.

In another embodiment, cannabis plant material can be suspended in asolvent and the mixture stirred for a defined period of time (e.g. 0-30sec or a reasonable length of time so as to suspend the material) beforebeing subjected to microwaves. In one embodiment, the defined period is30 seconds.

In another embodiment, cannabis material can be suspended in a solventand the mixture can be stirred while being subjected to microwaves. Inone embodiment, the defined period is 10 minutes and in anotherembodiment the defined period is 20 minutes. A table of workingmicrowave variables is provided below for reference.

TABLE 1 Total Pre- Power Holding Reaction Temperature time stirring Stirrate supplied Power Pressure (° C.) (mins) (sec) (rpm) (W) (W) (Bar) 15020 30 900 400 60 12 150 20 30 900 400 60 11 150 20 30 900 400 60 10 15020 30 900 400 55 13 150 20 30 900 400 60 13 150 20 30 900 400 60 12 17015 30 900 400 70 18 150 10 30 600 295 38 8.5 150 10 30 600 270 40 8.5150 10 30 600 360 40 9 150 10 0 600 400 75 8 150 10 0 600 400 75 8.5 15010 0 600 400 75 8.5 100 30 0 600 150 38 1 100 30 0 600 113 38 2 100 30 0600 145 30 1 150 10 30 600 265 60 8 150 10 30 600 255 38 9 150 10 30 600260 40 8.5 170 10 30 600 310 40 17 150 10 30 600 280 38 10 130 10 30 600250 30 5 100 5 30 600 400 30 2 100 5 30 600 390 40 2

The parameters below can be set by the user, depending on the type ofmicrowave equipment employed and the options available for usersettings:

-   -   Power (OFF)=Constant power or the maximum power applied when        heating the reaction mixture.    -   Initial Power (OFF)=Power applied initially when heating the        reaction mixture.    -   Fixed Hold Time (ON)=If ON, the time countdown starts when the        target temperature or target pressure is reached, i.e. the        initial time taken to reach the set temperature or pressure is        not included in the heating time.    -   Pressure (OFF)=Target pressure for reaction.    -   Cooling (OFF)=If OFF, cooling is not applied during the heating        process.    -   Absorption (NORMAL)=If NORMAL, power applied is initially        between 200 and 400 W, depending on the target temperature.    -   Temperature=Target temperature. (Note: The temperature is        monitored by an external infrared sensor that measures the        surface temperature of the glass vial in real time.)    -   Total time=Total time for all steps.    -   Pre-stirring=Stirring time before heating process.    -   Stir-rate=Rotational speed of magnetic stir bar.

The parameters below were observed and extrapolated.

Power supplied=Power used to achieve the target temperature.

Holding power=Power used to maintain the target temperature.

Reaction pressure=Maximum pressure during the reaction.

In some embodiments, the decarboxylated cannabinoid product can be useddirectly or further processed, purified or recovered prior to use.

Recovery 110

Optionally, after being subjected to microwaves, a preparation ofdecarboxylated cannabinoids can be recovered from the resultingsuspension at recovery step 110.

In one embodiment, extracted and decarboxylated cannabinoids arerecovered by filtering the solvent from the extract of cannabis plantmaterial to isolate the decarboxylated cannabinoids or decarboxylatedcannabinoid comprising fraction.

In another embodiment, extracted and decarboxylated cannabinoids arerecovered by filtering through an appropriate Celite® pad and/oractivated carbon (e.g. charcoal) to obtain clarified solution forsubsequent processing or use. In this embodiment, Celite® can be placedin a glass sintered funnel and then layered with activated carbon.Filtering agents can be washed with ethanol via vacuum filtration andextract can be dissolved in appropriate volume of suitable solvent suchas ethanol and transferred to a funnel. Vacuum can then be applied andthe filtering agent can be washed with the solvent until cannabinoidsare completely eluted. The resulting filtrate can then be concentratedto dryness (e.g. at 25° C.). Someone with skill in the art can alsoconceive employing functionalized membranes, cellulose filters or thelike to accomplish the above recovery task, instead of Celite® andactivated carbon pad.

Alternatively, the resulting preparation of decarboxylated cannabinoidsfrom step 108 can be collected and subsequently processed according toany of the extraction methods described in step 106, including but notlimited to sonication, reflux (Soxhlet) extraction and/or SFE.

Uses and Products

The decarboxylated cannabis resins can be used (i) directly as amedicine, natural health product, or for recreational use, or (ii) as araw material in the preparation of products, such as pharmaceutical,natural health products, or recreational use products for known uses ofdecarboxylated cannabinoid(s), such as known therapeutic or psychoactiveuses.

In one embodiment, the decarboxylated resin of the disclosure can beused directly, for example as a medicine or as a natural health product.

In one embodiment, a pharmaceutical composition can be formed comprisingthe resulting decarboxylated cannabinoid resin produced by the methodsdisclosed, which can further comprise a suitable pharmaceuticalacceptable carrier or excipient.

The pharmaceutical compositions of the present disclosure canadditionally comprise one or more further active pharmaceuticalingredients in addition to decarboxylated cannabinoids.

Pharmaceutical compositions can be prepared in various dosage formsdepending on the desired use and mode of administration, whether it isoral (e.g., tablet or liquid forms), a mist or other forms (aerosol,inhaler or intravenous suitable formulations), as desired, usingtechniques well known in the art.

Pharmaceutically acceptable carriers or excipients for various differentdosage forms are well-known in the art and include carriers, diluents,fillers, binders, lubricants, disintegrants, glidants, colorants,pigments, taste masking agents, sweeteners, flavorants, plasticizers,and any acceptable auxiliary substances such as absorption enhancers,penetration enhancers, surfactants, co-surfactants, and specializedoils. The proper excipient(s) is (are) selected based in part on thedosage form, the intended mode of administration, the intended releaserate, and manufacturing reliability. Examples of common types ofexcipients include various polymers, waxes, calcium phosphates, andsugars.

A person of skill in the art would reference known methods ofpharmaceutical preparations, such as described in Remington: The Scienceand Practice of Pharmacy, 22^(nd) Edition, Edited by Allen, Loyd V., Jr,2012.

The present disclosure is described in the following Examples, which areset forth to aid in the understanding of the disclosure, and should notbe construed to limit in any way the scope of the disclosure as definedin the claims which follow thereafter.

Examples

Experiment 1: Decarboxylated Cannabis Resin

Medicinal cannabis was subjected to extraction and chemical analysis byUPLC-MS.

Extraction Methodologies

FIG. 2A shows medical cannabis and decarboxylated cannabis resin. Fourtypes of extraction methods were performed as shown in the correspondingflow charts in FIG. 29. (A) Ultrasonication (B) Sohxlet extraction (C)Supercritical Fluid Extraction (Omar, J. Olivares, M. et al. J. Sep.Sci. 2013, 36, 1397-1404, incorporated herein by reference). (D) Closedsystem microwave extraction (in ethanol, hexane or isopropanol or liquidCO₂), as described in experiments 5-9 below.

UPLC-MS Methodology. Cannabinoid standards, cannabis extracts andcannabinoids in the donor samples were analyzed using Waters® ACQUITYUPLC H-Class System equipped with Quaternary Solvent Manager and SampleManager FTN. The detector used to monitor the samples was Waters® MS3100 mass spectrometer. Benzophenone, caffeine or Δ⁹-THC-d3 was used asan internal standard. Conditions are listed in Table 2.

TABLE 2 UPLC-MS Chromatographic conditions UPLC-MS Conditions Column:BEH (2.1 × 50 mm, C₁₈, 1.7 μm) Mass scan range (ESI +ve and −ve):150-500 m/z Flow rate: 0.6 mL/min Solvent: H₂O (0.1% formic acid;solvent A) MeOH (0.1% formic acid; solvent B) Gradient conditions: 0-4.5min: 30% A/70% B - 100% B 4.5-5.0 min: 100% B 5.0-5.2 min: 100% B - 30%A/70% B 5.2-6.0 min: 30% A/70% B

Extraction Results.

TABLE 3 Quantities of extracts and major cannabinoids obtained fromextraction Methods A-D of Strain I. Cannabinoid in resin (%) PlantExtract Total Δ⁹-THC Total CBD Method used (g) isolated (g) (THCA + THC)(CBDA + CBD) A 1.51 0.453 31.2 ± 0.5 66.1 ± 1.4 B 2.00 0.616 16.7 ± 0.233.3 ± 0.6 C 1.00 0.277 30.4 ± 0.9 59.3 ± 1.9 D 1.01 0.211 29.4 ± 7.633.8 ± 8.6

TABLE 4 Quantities of extracts and major cannabinoids obtained afterMethod D (closed system microwave extraction). Extract Cannabinoid inresin (%) Strain Plant used (g) isolated (g) Δ⁹-THC CBD I 1.01 ± 0.00030.211 ± 0.01  29.4 ± 7.6 33.8 ± 8.6 II 1.00 ± 0.0004 0.154 ± 0.004  1.9± 0.2 37.6 ± 4.1 III 1.00 ± 0.002  0.210 ± 0.009 49.8 ± 0.9 0

Chemical Analyses by UPLC-MS.

Mass spectra of Strain 1 before (FIG. 2b ) and after (FIG. 2c )decarboxylation of medical cannabis, indicates 100% conversion of CBDAand THCA into CBD and THC, respectively.

Closed system microwave extraction provides simultaneously extractionand decarboxylation of the cannabinoids, as observed during chemicalanalysis by UPLC-MS.

Experiment 2: Further Comparison of Various Extraction Methods,Decarboxylation of Cannabinoids and Production of Standard Curves

Method 1A: Ultrasonic Extraction (Sonication).

General Procedure:

-   -   1. Dried plant material was weighed and macerated using a mortar        and pestle.    -   2. Solvent was added (25-130 mL) and mixture sonicated for 5        mins at 25° C.    -   3. Solvent was decanted and filtered over a glass sintered        funnel using a vacuum filtration.    -   4. Steps 2 and 3 were repeated twice with the remaining fibre        material.    -   5. Filtrate was concentrated to dryness (at 25° C.) then weighed        (green resin).

Table 5 below shows the results of sonication of three common strains ofcannabis.

TABLE 5 Results of sonication extraction with various solvents on threestrains of cannabis. Solvent Isopropanol/ Strain Hexanes hexanes (1:1)Ethanol Cannabis strain 1 Plant 0.256  1.0013 0.246 (THC: 7.18/CBD: Used(g) 1.5084 8.6) Resin 0.0617 0.4748 0.1802 produced (g) 0.4531 Cannabisstrain 2 Plant N/A N/A 2.0018 (THC: 0/CBD: 9) Used (g) Resin N/A N/A0.4795 produced (g) Cannabis strain 3 Plant N/A 2.9475 (THC: 18.6/CBD:0) Used (g) Resin N/A N/A 1.1611 produced (g)

Method 1B: Filtration over Celite®/Activated Carbon.

Following Method 1A (described above), extracts were subjected tofiltration over Celite® and activated carbon in order to eliminate thegreen colour of extracts. The results are shown in Table 6.

General Procedure:

-   -   1. Celite® was placed in a glass sintered funnel, then layered        with activated carbon.    -   2. Extract was dissolved in 1 mL ethanol and transferred to        funnel.    -   3. Vial that contained extract was washed twice with 1.5 mL        ethanol and transferred to funnel.    -   4. Vacuum was then applied and filtering agent washed with        ethanol until filtrate was no longer UV active (60-70 mL).    -   5. Filtrate was concentrated to dryness (at 25° C.), then        weighed (orange resin).

TABLE 6 Results of extract filtration with Celite ®/Activated Carbon forthree strains of cannabis Strain Extract used (g) Extract isolated (g)Cannabis strain 1 0.2265 0.2095 (THC: 7.18/CBD: 8.6) Cannabis strain 20.2026 0.1717 (THC: 0/CBD: 9) Cannabis strain 3 0.4984 0.4561 (THC:18.6/CBD: 0)

Method 2: Soxhlet Extraction

General Procedure:

-   -   1. Dried plant material was weighed and macerated using a mortar        and pestle    -   2. Crushed material was then transferred to a cellulose        extraction thimble (43×123 mm; 2 mm thickness)    -   3. Thimble was then inserted into a large extractor (size:        55/50)    -   4. Solvent (400 mL) and stir bar were added to a round bottom        flask, which was then placed in the suitable DrySyn heating        block and connected to the extractor    -   5. Extractor was then connected to a large condenser (size:        55/50) and refluxing was done at 120° C. for 3.5 hrs    -   6. Once refluxing was complete, the solvent was concentrated to        dryness (at 25° C.) then weighed (green resin)

Table 7 below provides the results of Soxhlet extraction of cannabisaccording to the forgoing procedure.

TABLE 7 Results of Soxhlet extraction of cannabis. Strain Amount ofplant used (g) Extract isolated (g) Cannabis 2.0021 0.6163 (THC:7.18/CBD: 8.6)

Method 3: Supercritical Fluid Extraction (SFE)

General Procedure:

-   -   1. Dried plant material was weighed and macerated using a mortar        and pestle    -   2. Crushed plant material was transferred to a 10 mL extraction        vessel and subjected to either of the following conditions below    -   3. All fractions were combined and concentrated to dryness (at        25° C.) then weighed (green resin)

Method 3A: SFE Conditions

Solvent A = CO₂ Solvent B = ethanol Temperature = 40° C. BPR = 12 MPaPDA = 200-600 nm (monitoring at 254 nm) Acquisition time = 30 minsMethod time = 30.2 mins

-   -   i. 5 mins static with 1:1 A/B    -   ii. 25 mins dynamic with 1:1 A/B (Flow rate=10 mL/min; make up        pump=0.2 mL/min)    -   iii. Fractions were collected every 5 mins

Method 3B: SFE Conditions

Solvent A = CO₂ Solvent B = ethanol Temperature = 40° C. BPR = 12 MPaPDA = 200-600 nm (monitoring at 254 nm)

-   -   i. 15 mins dynamic with 100% A (Flow rate=10 mL/min)        -   Fractions were collected every 7.5 mins        -   Acquisition time=15 mins Method time=15.2 mins    -   ii. 30 mins dynamic with 80:20 A/B (Flow rate=10 mL/min; make up        pump=1 mL/min)        -   Fractions were collected every 5 mins        -   Acquisition time=30 mins Method time=30.2 mins

Table 8 below shows the results of SFE according to the conditionsoutlined in Methods 3A and 3B for cannabis.

TABLE 8 Results of SFE extraction of cannabis according to two sets ofconditions. Amount of Extract Strain Conditions plant used (g) isolated(g) Cannabis Method 3A 1.0047 0.2164 (THC: 7.18/CBD: 8.6) Method 3B1.0043 0.2561

Method 4: Microwave-Assisted Extractions with Ethanol (MAE) Followed bySFE.

Suitable conditions to promote decarboxylation of CBDA and THCA to CBDand THC, respectively, were determined with MAE.

The solvent used for this extraction was ethanol. However, since ethanolhas a boiling point of 78° C., the highest temperature that could beachieved when heating only ethanol in a sealed vessel under microwaveconditions had to be determined.

General Procedure:

-   -   1. Ethanol (11 mL) and a stir bar were placed in a 20 mL        microwave vial which was then sealed.    -   2. General microwave conditions:        -   a. Pre-stirring=30 secs        -   b. Run time=15 mins        -   c. Absorption=Normal

The results are shown in Table 9 below.

TABLE 9 Determination of highest temperature that could be achieved whenheating only ethanol in a sealed vessel under microwave conditions.Temperature Attempt # (° C.) Results 1 200 Maximum system pressureattained; Run aborted due to high pressure 2 150 Run completed 3 190Maximum system pressure attained; Run aborted due to high pressure 4 180Maximum system pressure attained; Run aborted due to high pressure 5 170Run completed

Once the maximum temperature that ethanol could be heated wasdetermined, the following conditions were performed with cannabis:

General Procedure:

-   -   1. Dried plant material was weighed and macerated using a mortar        and pestle    -   2. Crushed plant material was transferred to a 20 mL or 5 mL        microwave vial along with a stir bar    -   3. Ethanol (11 mL or 3 mL) was added to the vial such that the        plant material was completely submerged. The vial was then        sealed and subjected to the microwave conditions below:        -   a. Pre-stirring=30 secs        -   b. Run time=10 mins        -   c. Absorption=Normal    -   4. The suspension was filtered and the filtrate and plant fibre        collected separately    -   5. Filtrate was concentrated and plant fibre subjected to the        SFE conditions below:

Solvent A = CO₂ Solvent B = ethanol Temperature = 25° C. BPR = 12 MPaPDA = 200-600 nm (monitoring at 254 nm) Acquisition time = 20 minsMethod time = 20.2 mins

-   -   6. Gradient from 100% A to 50%; 0.1 mins-15 mins (Flow rate=10        mL/min; make up pump=1 mL/min)    -   7. Fractions were collected every 7.5 mins

The results are shown in Table 10 below.

TABLE 10 Extraction of cannabis by Microwave and SFE at variousmicrowave temperatures Extract Extract Temper- Amount isolated isolatedature of plant after after Strain Method (° C.) used (g) microwave (g)SFE (g) Cannabis spp. A 100 1.0012 0.3246 N/A (THC: 7.18/ B 130 0.25540.0592 0.0008 CBD: 8.6) C 150 0.2543 0.063 0.0005 D 170 0.2554 0.06440.0025

Table 14 (provided below) shows the analyses and quantification of thecannabinoids.

Method 5. SFE/Soxhlet/Sonication Extraction Followed by Microwave of theResin (for Decarboxylation).

General Procedure:

-   -   1. Resin isolated from Methods 1A (ethanolic extract), 2 and 3A        were dissolved in 3-3.5 mL ethanol and transferred to a 5 mL        microwave vial.    -   2. A stir bar was added and the vial sealed and subjected to        microwave conditions below:        -   a. Temperature=150° C.        -   b. Pre-stirring=30 secs        -   c. Run time=10 mins        -   d. Absorption=Normal    -   3. The reaction mixture was then concentrated.

The results are shown in Table 11 below.

TABLE 11 Weights of resins after subjecting to microwave heating. Resinisolated Amount of after Strain Method resin (g) microwave (g) Cannabisspp. 1A (Sonication) 0.2223 0.1692 (THC: 7.18/CBD: 8.6) 2 (Soxhlet) 0.2651 0.2211  3A (SFE; 50:50) 0.2164 0.1884

Chromatography Analyses (HPLC/MS/PDA)

The chromatographic profiles of the cannabis extracts were determined byLC-PDA-MS equipped with a Waters® 2545 binary gradient module LC,Waters® PDA2998 photodiode array detector (190-800 nm) and a Waters®3100 mass spectrometer (60-2000 Da).

LC was performed on an X-Bridge analytical C18 column (4.6 mm×150 mm, 5um I.D.) with 1.5 mL/min flow rate. Mass spectra were recorded using ESI(+ve) mode. The injection samples were filtered using Millex-GV® SyringeFilters (0.22 μm, EMD Millopore).

Chromatographic conditions were as follows:

-   -   Mobile phase:        -   A: Water/0.1% formic acid        -   B: Methanol/0.1% formic acid    -   Gradient:        -   0 to 25 min: 30% A/70% B→100% B        -   25 to 28 min: 100% B        -   28 to 30 min: 100% B→30% A/70% B        -   30 to 35 min: 30% A/70% B    -   Injection volume: 10 μL    -   Flow rate: 1.5 mL/min    -   Total run time: 35 min

UPLC/MS.

The cannabis extracts and cannabinoid standards were analyzed usingWaters® ACQUITY UPLC H-Class System equipped with Quaternary SolventManager, Sample Manager FTN, Acquity UPLC® BEH column (2.1×50 mm, C18,1.7 μm). The sample injection plate and the column were maintained at15° C. and 40° C., respectively. The detector used to monitor thesamples was Waters® MS 3100 mass spectrometer.

Chromatographic conditions were as follows:

Mobile phase:

-   -   A: Water/0.1% formic acid    -   B: Methanol/0.1% A formic acid    -   Gradient:    -   0 to 4.5 min: 30% A/70% B→100% B    -   4.5 to 5.0 min: 100% B    -   5.0 to 5.2 min: 100% B→30% A/70% B    -   5.2 to 6.0 min: 30% A/70% B    -   Injection volume: 2 μL    -   Flow rate: 0.6 mL/min    -   Total run time: 6 min

Standard Curves for Cannabinoids:

Standard cannabinoids samples were purchased from Cerilliant-CertifiedReference Standards in the form of 1.0 mg/mL solution in methanol.

1. Δ⁸-Tetrahydrocannabinol (Δ⁸-THC, Cat #. T-032, Lot FE10011501)

2. Δ⁹-Tetrahydrocannabinol (Δ⁹-THC, Cat #. T-005, Lot FE05271502)

3. Δ⁹-Tetrahydrocannabinolic acid A (THCA-A, Cat #. T-093, LotER02101506)

4. Δ²-Cannabidoil (CBD, Cat #. C-045, Lot FE012881502)

5. Cannabidiolic acid (CBDA, Cat #. C-144. Lot FE0181602)

6. Cannabinol (CBN, Cat #. C-046, Lot FE06081502)

The chemical structures of cannabinoids 1-6 are provided in FIG. 1.

Working stock solution of each standard sample was prepared usingwater/0.1% formic acid and methanol/0.1% formic acid. The finalconcentration of each stock sample was 50 μg/mL in 30% water/0.1% formicacid and 70% methanol/0.1% formic acid.

The stock samples (50 μg/mL) were diluted with mobile phase (30%water/0.1% formic acid and 70% methanol/0.1% formic acid) to obtain thefollowing concentrations:

0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, and 10.0 μg/mL

Not all the concentrations were included in the construction of thestandard curve. Some of the cannabinoids (e.g. 0.1 or 10.0 μg/mL) wereexcluded due to very low signal of saturation level.

Each concentration was run in triplicate. 2 μL injections were made andthe signal was recorded for up to 6 minutes. SIR+ve (311, 315, and 359m/z) or SIR −ve (313 and 357) and mass scan (150-500 m/z) in positivemode were monitored and recorded. SIR chromatograms were integrated andthe AUC was plotted vs. concentration (μg/mL).

TABLE 12 Cannabinoids standard curve - summary. Reference compoundRetention time (min) Range (μg/mL) Δ²-CBD 1.70  0.1-10.0 CBD-A 1.900.01-1.0  CBN 2.33 0.1-7.5 Δ⁹-THC 2.58 0.1-7.5 Δ⁸-THC 2.70  0.1-10.0THCA-A 3.42 0.01-1.0 

Standard curves for each of cannabinoids 1-6 as described above areprovided in FIGS. 4-9.

FIG. 4 shows a Standard Curve for Δ⁸-THC. Linear fit:y=10948149x+2153365, R=0.9984

FIG. 5 shows a Standard Curve for Δ⁹-THC. Linear fit:y=11609869x+2187215, R=0.9980.

FIG. 6 shows a Standard Curve for THCA. Linear fit: y=14550967x+119886,R=0.9992

FIG. 7 shows a Standard Curve for CBD. Linear fit: y=9601901x+1448932,R=0.9978.

FIG. 8 shows a Standard Curve for CBDA. Linear fit: y=9096880x+111409,R=0.9993.

FIG. 9 shows a Standard Curve for CBN. Linear fit: y=2595328x−397594,R=0.9967.

Table 13 provides the concentration (μg/mL) of cannabinoids in extractsobtained using microwave extraction method at different temperatures.The solid material (plant fiber) leftover after the microwave reactionwas exposed to SFE extraction (method 3A). The total volume of eachmicrowave reaction was 3 mL.

TABLE 13 Concentration (μg/mL) of cannabinoids in extracts obtainedusing microwave extraction method at different temperatures Extractionconditions Δ⁸-THC Δ⁹-THC THCA CBD CBN Microwave, 100° C., 10 min; 0 554358 500 252 No SFE done for this sample N/A N/A N/A N/A N/A Microwave,130° C., 10 min 0 7046 225 7565 443 SFE 0 243 0 302 32 Microwave, 150°C., 10 min 0 7201 0 9408 508 SFE 0 86 0 135 82 Microwave, 170° C., 10min 0 6502 0 8566 495 SFE 0 936 0 1321 120

FIG. 10 shows the amount of Δ⁹-THC and THC-A present in samplesextracted using microwave method at different temperatures. The amountof each form of THC is shown as a percentage of total amount of THC(neutral and acidic form).

Table 14 shows the amount of cannabinoids in the cannabis extracts,after subjecting to Method 5. See also Table 11. Note: CBN appears to beformed during the microwave based decarboxylation of THCA. CBDA was notquantified.

TABLE 14 Amount of cannabinoids in the cannabis extracts, aftersubjecting to Method 5 Δ⁸-THC Δ⁹-THC Δ⁹-THCA-A Δ²-CBD CBN mg/g mg/g mg/gmg/g mg/g Sonication 0 0 69.47 4.93 0 (Method 1A) Soxhlet alone 0 44.3240.71 25.4 0 (Method 2) SFE alone 0 1.91 36.91 3.68 0 (Method 3A)Sonication + μw 0 107.18 0 145.1 9.53 (Method 1A and 5) Soxhlet + μw 0113.98 0 161.75 10.08 (Method 2 and 5) SFE + μw 0 96.48 0 122.66 8.96(Method 3A and 5) μW = microwave ND = not determined

A plot of the above data is shown in FIGS. 11 and 12, wherein FIG. 11illustrates concentrations in the extracts using EtOH, Soxhlet and SFE,without subjecting cannabis to microwave conditions and FIG. 12 showsthe concentrations of cannabinoids first extracting cannabis using EtOHor Soxhlet or SFE followed by microwave heating.

Table 15 shows the yield (mg/g of plant material) of cannabinoids in theextracts obtained using microwave extraction method at differenttemperatures. The solid material (plant fiber) left over after themicrowave reaction was exposed to SFE extraction (method 3A). The totalvolume of each microwave reaction was 3 mL. CBDA was not quantified.

TABLE 15 Yield (mg/g of plant material) of cannabinoids in extractsobtained using microwave extraction method at different temperatures Δ⁸-Δ⁹- THC THC THCA-A CBD CBN Conditions mg/g mg/g mg/g mg/g mg/g Microwave100° C., 10 min 0 45 29  41 3 No SFE N/A N/A N/A N/A N/A Microwave, 130°C., 10 min 0 83 3 89 5 Follow-up SFE 0 3 0 4 0 Microwave, 150° C., 10min 0 85 0 111 6 Follow-up SFE 0 1 0 2 1 Microwave, 170° C., 10 min 0 760 101 6 Follow-up SFE 0 11 0 16 1

FIG. 13 shows cannabinoids obtained from cannabis after microwaveheating only.

FIG. 14 shows microwave followed by SFE extraction. The cannabinoidsfrom microwave solvent and SFE extractions added together.

FIG. 15 shows various cannabinoid concentrations from cannabis aftermicrowave heating followed by SFE extraction. In FIG. 15, thecannabinoids from both extractions have been added together. The acidicand neutral form of THC were added together to represent the total THCextracted by each method.

FIG. 16 shows a chromatogram of mass scan (150-500 m/z) recorded inpositive mode of ESI using Waters® MS3100 mass detector. The sample wasobtained from microwave extraction at 170° C., 15 min. The arrows pointto the retention times of CBD, CBDA, Δ⁹-THC, and THCA. Due to the lowsample concentration the peaks are not visible in this chromatogram.Single ion recording (SIR+ve) obtained for this sample shows theindividual peaks representing CBD and Δ⁹-THC (FIG. 17), CBDA and THCA(FIG. 18). If an analyte is absent or not detected in this mode, theexpected retention time is shown

FIG. 17 shows a Single Ion Recording (SIR+ve, m/z=315) to detectCannabidiol (CBD) and Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) in a sampleobtained from microwave extraction at 170° C., 15 min (mass scan shownin FIG. 16).

FIG. 18 shows a Single Ion Recording (SIR+ve, m/z=359) to detectCannabidiolic acid (CBDA) and Tetrahydrocannabinolic acid (THCA) in asample obtained from microwave extraction at 170° C., 15 min (mass scanshown in FIG. 16). Positive mode (ESI+ve) is not a favorable one for thedetection of the acidic forms of cannabinoids.

FIG. 19 shows a chromatogram of mass scan (150-500 m/z) recorded innegative mode of ESI using Waters® MS3100 mass detector. The sample wasobtained from microwave extraction at 170° C., 15 min. The arrows pointto the retention times of CBD, CBDA, Δ⁹-THC, and THCA. Due to the lowsample concentration the peaks are not visible in this chromatogram.Single ion recording (SIR −ve) obtained for this sample shows theindividual peaks representing CBD and Δ⁹-THC (FIG. 20), CBDA and THCA(FIG. 21). If an analyte is absent or not detected in this mode, theexpected retention time is shown.

FIG. 20 shows a Single Ion Recording (SIR −ve, m/z=313) to detectCannabidiol (CBD) and Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) in a sampleobtained from microwave extraction at 170° C., 15 min (mass scan shownin FIG. 19). The neutral forms of cannabinoids are not easily detectablein the negative mode therefore no peaks observed as compared to thepositive mode (see FIG. 17).

FIG. 21 shows a Single Ion Recording (SIR −ve, m/z=357) to detectCannabidiolic acid (CBDA) and Tetrahydrocannabinolic acid (THCA) in asample obtained from microwave extraction at 170° C., 15 min (mass scanshown in FIG. 19). ESI −ve mode is useful for the detection of theacidic forms of cannabinoids.

FIG. 22 shows a chromatogram of mass scan (150-500 m/z) recorded inpositive mode of ESI using Waters® MS3100 mass detector. The sample wasobtained from microwave extraction at 130° C., 10 min. Retention timesof CBD, CBDA, Δ⁹-THC, and THCA are shown. Single ion recording (SIR+ve)obtained for this sample shows the individual peaks representing CBD andΔ⁹-THC (FIG. 23) and CBDA and THCA (FIG. 24). If an analyte is absent ornot detected in this mode, the expected retention time is shown.

FIG. 23 shows a Single Ion Recording (SIR+ve, m/z=315) to detectCannabidiol (CBD) and Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) in a sampleobtained from microwave extraction at 130° C., 10 min (mass scan shownin FIG. 22).

FIG. 24 shows a single Ion Recording (SIR+ve, m/z=359) to detectCannabidiolic acid (CBDA) and Tetrahydrocannabinolic acid (THCA) in asample obtained from microwave extraction at 130° C., 10 min (mass scanshown in FIG. 22). Due to relatively high concentration of the analyzedsample the acidic forms of the CBD and THC are detected by the negativemode.

FIG. 25 shows a chromatogram of mass scan (150-500 m/z) recorded innegative mode of ESI using Waters® MS3100 mass detector. The sample wasobtained from microwave extraction at 130° C., 10 min. Retention timesof CBD, CBDA, Δ⁹-THC, and THCA are shown. Single ion recording (SIR −ve)obtained for this sample shows the individual peaks representing CBD andΔ⁹-THC (FIG. 26) and CBDA and THCA (FIG. 27). If an analyte is absent ornot detected in this mode, the expected retention time is shown.

FIG. 26 shows a Single Ion Recording (SIR −ve, m/z=313) to detectCannabidiol (CBD) and Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) in a sampleobtained from microwave extraction at 130° C., 10 min (mass scan shownin FIG. 25). Neutral cannabinoids (CBD and Δ⁹-THC are not visible in thenegative mode detection as compared to the positive mode (FIG. 23).

FIG. 27 shows a Single Ion Recording (SIR −ve, m/z=357) to detectCannabidiolic acid (CBDA) and Tetrahydrocannabinolic acid (THCA) in asample obtained from microwave extraction at 130° C., 10 min (mass scanshown in FIG. 25). Some CBDA and THCA still present showing partialdecarboxylation.

Summary of Results for Experiment 2

FIGS. 4-9 provide standard chromatography curves for Δ⁸-THC, Δ⁹-THC,THC-A, CBD, CBDA and CBN, respectively. These standard curves weregenerated for concentrations between 0 and 10 μg/mL and subsequentlyused for determining the concentration of each compound in variousproducts formed using the embodiments described herein.

Table 5 shows the use of various solvents during ultrasonic extraction.There were also minimal amounts of cannabis plant material lost duringthe recovery filtration process (see Table 6). This also appears to besimilar recovery across different extraction methods—solvent, Soxhlet,SFE extraction (See Tables 7 and 8).

When conducting cannabinoid extraction/decarboxylation in ethanol usinga microwave, due to the boiling point of ethanol, it was shown thatextraction/decarboxylation of cannabinoids using a microwave is bestconducted at temperatures below 180° C., for example at 160° C.±10° C.(e.g. +/−the acceptable standard of error). It was also shown under theconditions used that conversion of THCA to THC (the desireddecarboxylated product) was better at 130° C. than at 100° C., and thatif conducted using microwave alone versus microwave and SFE,temperatures above 130° C., for example from 150° C. to 170° C. showedmore efficient conversion. (See Tables 13 and 15 as well as FIG. 10).However, there appears to be significant loss of material if followed bySFE (see Tables 9 and 10, and 15). As such, the least number of stepsand processes to obtain the desired results is the one-stepextraction/decarboxylation method.

FIGS. 11 and 12 and Table 14 show that extraction resulted in minimal orno decarboxylated THC product, while the addition of the microwave stepresulted in a significant increase in decarboxylated THC.

FIG. 13 shows concentrations of Δ⁹-THC, THC-A, CBD and CBN from cannabisafter microwave radiation only at temperatures ranging between 100° C.and 170° C. FIG. 13 shows that microwave radiation at temperaturesbetween 130° C. and 170° C. were optimal when ethanol was used as thesolvent.

FIG. 14 shows concentrations of Δ⁹-THC, THC-A, CBD and CBN from thecannabis plant material after microwave radiation and subsequent SFEextraction at temperatures ranging between 100° C. and 170° C. Use ofSFE extraction does not appear to significantly increase yield ofdecarboxylated cannabinoids.

FIG. 15 shows concentrations of total THC, CBD and CBN from the cannabisafter microwave radiation and subsequent SFE extraction at atemperatures ranging between 100° C. and 170° C., where the acidic andneutral form of THC are added together to represent the total THCextracted by each method. Again, use of SFE extraction does not appearto significantly increase yield of decarboxylated cannabinoids.

From the above results, it appears that the significant decarboxylatedproduct THC results from microwave extraction and that the addition of asecond extraction step, such as SFE, does not appear to change thecannabinoid profile. It further shows that CBD as well as THC componentsare present in extract after microwave extraction alone or withmicrowave and SFE extractions combined.

In summary, the present disclosure shows that extraction anddecarboxylation of cannabis plant material can be done concurrentlyusing a microwave, set at a temperature below the boiling point of theextraction solvent, such as ethanol, without the need for a separateextraction step. This optimizes decarboxylated cannabinoid formation andrecovery and can produce a more consistent and reproducible product withconsistent and reproducible efficacy and therapeutic results.

An extraction step can also be included before use of a microwave.Although an extraction step after the microwave step is possible, it isnot necessary.

Experiment 3: Supercritical Fluid Extraction (SFE)

General Procedure:

-   -   1. Dried plant material was weighed and macerated using a mortar        and pestle    -   2. Crushed plant material was transferred to a 10 mL extraction        vessel and subjected to either of the following conditions below    -   3. Fractions from each run were collected every 5 mins and        combined and concentrated to dryness (at 25° C.) then weighed        (green resin)    -   4. Extraction was done three times with same plant fibre

A) SFE conditions

Solvent A = CO₂ Solvent B = ethanol Temperature = 25° C. BPR = 12 MPaPDA = 200-600 nm (monitoring at 254 nm)

Flow rate=10 mL/min; make up pump=1 mL/min

Acquisition time=30 mins Method time=30.2 mins

i. 0.1 min-25 mins; gradient of 0-50% B in A

26 mins; 100% A

30 mins; 100% A

Table 16 below shows the corresponding results.

TABLE 16 Weights of extract after SFE. Strain Run Extract isolated (g)Variety 1 = 1.0023 g 1 0.2634 (THC: 7.18/CBD: 8.6) 2 0.0038 3 0.010

Experiment 4: Microwave-Assisted Decarboxylation of Extract with Ethanol(MAE)

General Procedure:

-   -   1. Extract isolated from the SFE method described above was        dissolved in 5-10 mL ethanol and an appropriate volume        transferred to 5 mL microwave vials    -   2. Added additional volume of ethanol and a stir bar to the 5 mL        microwave vials    -   3. The vials were sealed and subjected to one of two microwave        conditions below:        -   (a) Temperature=150° C.; run time=10 mins; stir rate=600            rpm; absorption=Normal        -   (b) Temperature=100° C.; run time=30 mins; stir rate=600            rpm; absorption=Normal    -   4. The solution was then concentrated at 35° C. after        transferring to 20 mL vials

The results are shown in Table 17 below.

TABLE 17 Weights of the resins after subjecting to microwave heating.Amount of Extract resin used isolated after Strain Method (g) microwave(g) Variety 1 (a) 0.0832 0.0212 (THC: 7.18/CBD: 8.6) (b) 0.0832 0.0612(a) 0.2095 0.1589 Variety 2 (a) 0.1693 0.1114 (THC: 0/CBD: 9) Variety 3(a) 0.2722 0.1759 (THC: 18.6/CBD: 0)

Experiment 5: Additional Microwave-Assisted Extractions with Ethanol(MAE), Optimization of Conditions for Decarboxylation

General Procedure:

-   -   1. Dried plant material was weighed and macerated using a        laboratory blender at 22,000 rpm for 60 secs    -   2. Crushed plant material was re-weighed and transferred to a 20        mL microwave vial along with a stir bar    -   3. Ethanol (10 mL) was added to the vial which was then sealed        and subjected to the microwave conditions below:        -   (a) Temperature=170° C.; run time=15 mins; pre-stirring=30            sec; stir rate=900 rpm; absorption=Normal        -   (b) Temperature=150° C.; run time=20 mins; pre-stirring=30            sec; stir rate=900 rpm; absorption=Normal    -   4. The suspension was filtered and the filtrate and plant fibre        collected separately    -   5. Filtrate was then concentrated at 35° C., then transferred to        a 20 mL vial using ethanol and again concentrated at 35° C.,        then stored in the refrigerator

The results are shown in Table 18 below.

TABLE 18 Amount of Amount of plant plant Extract before after isolatedmac- mac- after eration eration microwave Strain Method (g) (g) (g)Variety 1 (a) 1.0062 0.8256 0.2265 (THC: 7.18/CBD: 8.6) Variety 2 (a)1.0042 0.8382 —^(a) (THC: 0/CBD: 9) (b) 1.0050 0.8293 0.2155 Variety 3(b) 1.0063 0.7883 0.1166 (THC: 18.6/CBD: 0) ^(a)Desired temperaturecould not be achieved due to pressure build-up; cap of vial popped offcausing solvent and plant fibre to escape from vial.

Since condition (b) in step 3 above proved successful at that scale,subsequent decarboxylations were performed using that microwavecondition and were done in triplicate. However, the followingmodifications were made:

Step 1: Blend at 18,000 rpm for 4 secs

Step 4: Filtration done over celite/activated carbon (as previouslydescribed in an earlier report)

Step 5: Filtrate was then concentrated at 35° C., weighed, transferredto a 20 mL vial using ethanol, then stored in the refrigerator

The results are shown in Table 19 below.

TABLE 19 Amount Amount of plant of plant Average before after Extractmac- mac- Extract isolated eration eration isolated (g) ± Strain Runs(g) (g) (g) SD Variety 1 1 1.0079 0.9260 0.2044 0.2105 ± (THC: 7.18/CBD:8.6) 2 1.0084 0.9594 0.2184 0.007 3 1.0080 0.9254 0.2088 Variety 2 11.0032 0.8287 0.1624 0.1654 ± (THC: 0/CBD: 9) 2 1.0038 0.8467 0.17210.006 3 1.0040 0.8585 0.1617 Variety 3 1 1.0017 0.9169 0.2225 0.2212 ±(THC: 18.6/CBD: 0) 2 1.0054 0.8952 0.2068 0.014 3 1.0033 0.9021 0.2343

This experiments shows the consistency of the extraction method.

Experiment 6: Cannabis Extractions (1 Gram Scale)

General Procedure (˜1.0 g Batch; Before Maceration):

-   -   1. Dried plant material was weighed and macerated using a        laboratory blender at 18,000 rpm for 4 secs    -   2. Crushed plant material was re-weighed (0.875 g) and        transferred to a 20 mL microwave vial along with a stir bar    -   3. Ethanol (10 mL) was added to the vial which was then sealed        and subjected to the microwave conditions below:    -   Temperature=150° C.; run time=20 mins; pre-stirring=30 sec; stir        rate=900 rpm; absorption=Normal    -   4. The suspension was filtered over celite/activated carbon and        the filtrate and plant fibre collected separately    -   5. Filtrate was then concentrated at 35° C., then transferred to        a 20 mL vial using ethanol and again concentrated at 35° C.,        then stored in the refrigerator    -   6. Decarboxylations were performed in triplicate

Winterization Procedure:

-   -   1. Resin was dissolved in ethanol (10 mL/g) and heated at 40° C.        for 5 mins in a water bath    -   2. Vial containing extract solution was cooled to −75° C. using        a dry ice/acetone bath for 3-4 hrs    -   3. Solution was filtered with a pre-weighed syringe filter in 20        mL vials Filter specifications: Millex®—GV (sterile), Low        Protein Binding Durapore (PVDF) Membrane; 0.22 μm pore size; 33        mm diameter    -   4. Filter was washed with ethanol that had been cooled for 5        mins at −75° C. using a dry ice/acetone bath    -   5. Filtrate was concentrated at 35° C. and extract was weighed    -   6. Syringe filter was weighed after 2-3 days drying in the        fumehood

TABLE 20 The quantities of cannabis plants used and the amounts ofextract obtained before and after winterization. Extract Extract PlantPlant isolated isolated before after before after maceration macerationwinter- winter- Strain Runs (g) (g) ization (g) ization (g) Variety 1 11.008 ± 0.9369 ± 0.2105 ± 0.1962 ± (THC: 7.18/ 2 0.0003 0.019 0.0070.007 CBD: 8.6) 3 Variety 2 1 1.004 ± 0.8446 ± 0.1654 ± 0.1541 ± (THC:0/ 2 0.0004 0.015 0.006 0.004 CBD: 9) 3 Variety 3 1 1.003 ± 0.9047 ±0.2212 ± 0.2095 ± (THC: 18.6/ 2 0.0019 0.011 0.014 0.009 CBD: 0) 3

TABLE 21 The quantities of cannabinoids (as % of resin and mg/g ofplant) in the extracts isolated, before and after winterization.Cannabinoid in resin before Cannabinoid in resin after winterizationwinterization Δ⁹- Δ⁹- Δ⁹- Δ⁹- CBD CBD THC THC CBD CBD THC THC StrainsRuns (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) Variety 1 1 n.d. n.d.n.d. n.d. n.d. n.d. n.d. n.d. (THC: 2 7.18/CBD: 3 8.6) Variety 2^(b) 1n.d. n.d. 37.6 ± 4.1 67.1 ± 7.1 1.9 ± 0.1 3.5 ± 0.2 (THC: 2^(a) 0/CBD:9) 3^(a) Variety 3^(b) 1 n.d. n.d. 49.8 ± 0.8  119.0 ± 1.4  (THC: 2^(a)18.6/CBD: 0) 3^(a) n.d. = not determined; samples were used up beforere-analysis with internal standard was performed. ^(a)Averagecalculations based on runs 2 and 3 only.Summary of results: Extracting and decarboxylating 1 gram scale batch ofcannabis was successful.

Experiment 7: Larger Scale MAE (˜3.75 g Batch; Before Maceration) Usingthe Modified Conditions

-   -   1. Dried plant material was weighed (˜3.75 g per batch) and        macerated using a laboratory blender at 18,000 rpm for 4 secs.    -   2. Crushed plant material was re-weighed and transferred to 3×20        mL microwave vials along with stir bars (˜1.2 g of plant fibre        per vial)    -   3. 95%-100% Ethanol (12 mL) was added to the vial which was then        sealed and subjected to the microwave conditions below:    -   Temperature=150° C.; run time=30 mins; pre-stirring=30 sec; stir        rate=900 rpm; absorption=Normal    -   Note: Time for decarboxylation was increased to 30 mins since        analysis revealed that decarboxylation was incomplete at that        scale    -   4. The 3×20 mL vials were combined after decarboxylation and the        suspension was filtered and the filtrate and plant fibre        collected separately    -   5. Filtrate was then concentrated at 35° C., then transferred to        a 20 mL vial using ethanol and again concentrated at 35° C.,        weighed and stored in the refrigerator    -   6. Decarboxylations were performed in duplicate

The results are shown in Table 22 below.

TABLE 22 Amount Amount of plant of plant Average before after Extractmac- mac- Extract isolated eration eration isolated (g) ± Strain Runs(g) (g) (g) SD Variety 1 1 3.7922 3.6268 0.9967 0.9437 ± (THC: 7.18/CBD:8.6) 2 3.7228 3.5800 0.8906 0.075 Variety 2 1 —^(b) 1.2427 —^(c) —^(c)(THC: 0/CBD: 9) Variety 3 1 3.7100 3.5978 1.0658 1.1572 ± (THC:18.6/CBD: 0) 2 3.8012 3.6035 1.2486 0.1293 ^(b)Plant fibre is suppliedas pulverized buds. Size was appropriate, therefore no furthermaceration was done. ^(c)Desired temperature of 150° C. could not beachieved due to pressure build-up.

-   -   Summary of results: From the results above, it can be concluded        that larger scale microwave assisted extraction was successful,        at 3-4 grams scale. This method can be scaled up into multi-gram        and larger scales with appropriate adjustments to conditions.

Experiment 8: Large Scale Microwave-Assisted Extractions with Ethanol(MAE)

(A) General Procedure (˜3.75 g Batch; Before Maceration):

-   -   1. Dried plant material was weighed and macerated using a        laboratory blender at 18,000 rpm for 4 secs    -   2. Crushed plant material was re-weighed and transferred to 3×20        mL microwave vials along with stir bars (˜1.2 g of plant fibre        per vial)    -   3. 95%-100% Ethanol (12 mL) was added to the vial which was then        sealed and subjected to the microwave conditions below:        -   Temperature=150° C.; run time=30 mins; pre-stirring=30 sec;            stir rate=900 rpm; absorption=Normal        -   Note: Time for decarboxylation was increased to 30 mins            since analysis revealed that decarboxylation was incomplete            at that scale    -   4. All 20 mL vials were combined after decarboxylation and the        suspension was filtered and the filtrate and plant fibre        collected separately (first batch)    -   5. Filtrate was then concentrated at 35° C., then transferred to        a 20 mL vial using ethanol and again concentrated at 35° C.,        weighed and stored in the refrigerator    -   6. Decarboxylations were performed in duplicate

Winterization Procedure:

1. Same as previously described

TABLE 23 The quantities of cannabis plants used and the amounts ofextract obtained before and after winterization. Plant Extract isolatedExtract before Plant after before isolated after maceration macerationwinterization winterization Strain Runs (g) (g) (g) (g) Variety 1 1^(a)3.7575 ± 0.049 3.6034 ± 0.033 0.9967 0.9437 ± 0.075 0.7794 (THC: 20.8906 7.18/CBD: 8.6) Variety 2 1 1.2427 ^(b) n.d. n.d n.d n.d (THC:0/CBD: 9) Variety 3 1 3.7556 ± 0.064 3.6007 ± 0.004 1.0618 1.1543 ±0.131 0.7984 0.8973 ± 0.140 (THC: 2 1.2468 0.9961 18.6/CBD: 0) n.d. =not determined. Desired temperature of 150° C. could not be achieved dueto pressure build-up; nothing further was done with buds. ^(a)Nowinterization was performed on run 1; winterization done on run 2 only.^(b) Plant fibre is supplied as pulverized buds. Size was appropriate,therefore no further maceration was done.

TABLE 24 The quantities of cannabinoids (as % of resin and mg/g ofplant) in the extracts isolated, before and after winterization.Cannabinoid in resin before Cannabinoid in resin after winterizationwinterization Δ⁹- Δ⁹- Δ⁹- Δ⁹- CBD CBD THC THC CBD CBD THC THC StrainsRuns (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) Variety 1 1 n.d. n.d.n.d. n.d. 39.0 ± 2.2 84.9 ± 4.7 30.3 ± 1.4 66.1 ± 3.0 (THC: 2^(a)7.18/CBD: 8.6) Variety 3 1 68.6 ± 8.4 219.0 ± 2.7 61.1 ± 4.6 153.2 ±35.0 (THC: 2 18.6/CBD: 0) n.d. = not determined; samples were used upbefore re-analysis with internal standard was performed.^(a)Calculations based on run 2 only.

(B) General Procedure (˜7.5 q Batch; Before Maceration):

-   -   1. Dried plant material was weighed and macerated using a        laboratory blender at 18,000 rpm for 10 secs    -   2. Crushed plant material was re-weighed and transferred to 6×20        mL microwave vials along with stir bars (˜1.2 g of plant fibre        per vial)    -   3. See steps 3-6 in (A) above

Winterization Procedure:

1. Same as previously described

TABLE 25 The quantities of Variety 1 (THC: 7.18/CBD: 8.6) used and theamounts of extract obtained before and after winterization. Amount ofAmount of Extract Extract plant before plant after isolated beforeisolated after maceration maceration winterization winterization Runs(g) (g) (g) (g) 1 7.5361 3.6156 ± 1.0154 ± 0.8115 ± 2 0.002 0.064 0.0303 7.5063 3.6640 ± 0.9327 ± 0.8247 ± 4 0.026 0.014 0.022

TABLE 26 The quantities of cannabinoids (as % of resin and mg/g ofplant) in the Variety 1 extract isolated, before and afterwinterization. Cannabinoid in resin after Cannabinoid in resin beforewinterization winterization Δ⁹- CBD CBD Δ⁹-THC Δ⁹-THC CBD CBD THC Δ⁹-THCRuns (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) 1 40.6 ± 3.9 113.7 ±3.7 31.1 ± 2.0 87.3 ± 0.1 31.6 ± 5.5 70.8 ± 9.7 23.1 ± 4.1 51.6 ± 7.3 23 29.2 ± 0.1  74.3 ± 0.9 20.9 ± 1.0 53.3 ± 3.0 44.0 ± 2.9 99.0 ± 4.731.3 ± 0.7 70.3 ± 0.2 4

(C) General Procedure (˜4.0 and 7.0 g Batches; Before Maceration):

-   -   1. Dried plant material was weighed and macerated using a        laboratory blender at 18,000 rpm for 10 secs    -   2. Crushed plant material was re-weighed and transferred to 20        mL microwave vials along with stir bars (˜1.2 g of plant fibre        per vial) 13. For ˜4.0 g batch, see steps 3-5 in (A) above    -   4. For ˜7.0 g batch, see steps 3-5 in (A) above

Winterization Procedure:

1. Same as previously described

TABLE 27 The quantity of Variety 1 plant (THC: 7.18/CBD: 8.6) used andthe amount of extract obtained before and after winterization. Amount ofAmount of Extract Extract plant before plant after isolated beforeisolated after maceration maceration winterization winterization Runs(g) (g) (g) (g) 1 4.0325 3.8199 0.8889 0.7358 2 7.1293 6.8717 1.52441.4101

TABLE 28 The quantities of cannabinoids (as % of resin and mg/g ofplant) in the Variety 1 extract isolated, before and afterwinterization. Cannabinoid in resin after Cannabinoid in resin beforewinterization winterization Δ⁹- CBD CBD Δ⁹-THC Δ⁹-THC CBD CBD THC Δ⁹-THCRuns (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) 1 30.2 ± 0.0 70.3 ± 0.127.1 ± 0.4 63.0 ± 0.0 38.4 ± 6.1 73.9 ± 11.8 32.1 ± 7.5 61.8 ± 14.4 233.6 ± 2.9 74.5 ± 6.5 28.9 ± 3.2 64.2 ± 7.1 42.5 ± 0.5 87.2 ± 1.1  37.0± 2.9 75.9 ± 5.9 

-   -   Summary of results: Extracting and decarboxylating 3.75 gram        scale batch of cannabis was successful.

Experiment 9: Larger Scale Microwave-Assisted Extractions with Ethanol(MAE) and Winterization

-   -   1. Dried plant material was weighed (˜7.5 g per batch) and        macerated using a laboratory blender at 18,000 rpm for 10 secs    -   2. Crushed plant material was re-weighed and transferred to 6×20        mL microwave vials along with stir bars (˜1.2 g of plant fibre        per vial)    -   3. 95%-100% Ethanol (12 mL) was added to the vial which was then        sealed and subjected to the microwave conditions below:        -   Temperature=150° C.; run time=30 mins; pre-stirring=30 sec;            stir rate=900 rpm; absorption=Normal    -   4. The 3×20 mL vials were combined after decarboxylation and the        suspension was filtered and the filtrate and plant fibre        collected separately    -   5. Filtrate was then concentrated at 35° C., then transferred to        a 20 mL vial using ethanol and again concentrated at 35° C.,        weighed and stored in the refrigerator    -   6. Resin was dissolved in ethanol (10 mL/g)    -   7. Vials containing extract solution were cooled to −75° C.        using a dry ice/acetone bath for 4 hrs    -   8. Solution was filtered with a pre-weighed syringe filter in 20        mL vials        -   Filter specifications: Millex®—GV (sterile), Low Protein            Binding Durapore® (PVDF) Membrane, 0.22 μm pore size, 33 mm            diameter    -   9. Filter was washed with ethanol that had been cooled for 5        mins at −75° C. using a dry ice/acetone bath    -   10. Filtrate was concentrated at 35° C. and vacuum dried for 2        days at 40° C. (via water bath), then extract was weighed    -   11. Syringe filter was weighed after 2-3 days drying in the        fumehood

The results are shown in Table 29 below.

TABLE 29 Amount of mg of plant Amount of cannabinoid/ before plant afterExtract g of maceration maceration isolated plant (%) Strain Runs (g)(g) (g) THC CBD Variety 1 1 7.5063 3.682 0.8403 7.0 10.7 (THC: 2 3.64590.8091 7.7 12.2 7.18/CBD: 8.6)

-   -   Summary of results: 7.5 g large scale batch of cannabis        extraction and decarboxylation was successful. These experiments        demonstrate that the methods of the disclosure consistently        extract and decarboxylate cannabinoids and can be used on a        commercial scale.

Experiment 10: Winterization

Winterization is a procedure typically used to remove waxes and otherpartially soluble materials at 0±10° C. temperature range. This processmay not be applicable, if there are no waxes present in the extract, orsuch hydrophobic molecules are broken down, and would be solidify at theice-bath or below-zero temperatures.

Experiment 10A

To remove waxes, the solutions of extract which had been stored in therefrigerator (˜5° C.) for 1-3 weeks were manipulated as follows:

-   -   1. Solution was filtered using a syringe filter in 20 mL vials        -   Filter specifications: Millex®—GV (sterile)            -   Low Protein Binding Durapore (PVDF) Membrane            -   0.22 μm pore size            -   13 m diameter    -   2. Filtrate was concentrated at 35° C. and vacuum dried for 3        days at 40° C. (via water bath)    -   3. Extracts were then re-weighed

Experiment 10E3

-   -   1. Resin was dissolved in 95%-100% ethanol (10 mL/g) and heated        at 40° C. for 5 mins in a water bath    -   2. Vial containing extract solution was cooled to −75° C. using        a dry ice/acetone bath for 3-3.75 hrs    -   3. Solution was filtered with a pre-weighed syringe filter in 20        mL vials (see above for filter specifications); filter was        washed with ethanol that had been cooled for 5 mins at −75° C.        using a dry ice/acetone bath    -   4. Filtrate was concentrated at 35° C. and extract was weighed    -   5. Syringe filter was weighed after 2-3 days drying in the        fumehood

The results are shown in Tables 30a and b below.

TABLE 30a Amount of extract isolated before and after winterizationmethods After 1^(st) After 2^(nd) Before 1^(st) winterizationwinterization winterization and drying and drying Strain Runs (g) (g)(g) Variety 1 1 0.2044 0.2111 0.2031 (THC: 7.18/CBD: 8.6) 2 0.21840.2345 0.1964 3 0.2088 0.1981 0.1891 Variety 2 1 0.1624 0.1677 0.1580(THC: 0/CBD: 9) 2 0.1721 0.1622 0.1508 3 0.1617 0.1677 0.1536 Variety 31 0.2225 0.2088 0.1996 (THC: 18.6/CBD: 0) 2 0.2068 0.2221 0.2145 30.2343 0.2243 0.2145

TABLE 30b Average extract isolated before and after winterizationmethods After 1st After 2nd Before 1st winterization winterizationwinterization and drying and drying Strain Runs (g) (g) (g) Variety 1 10.2105 ± 0.2146 ± 0.1962 ± (THC: 7.18/CBD: 8.6) 2 0.007 0.018 0.007 3Variety 2 1 0.1654 ± 0.1659 ± 0.1541 ± (THC: 0/CBD: 9) 2 0.006 0.0030.004 3 Variety 3 1 0.2212 ± 0.2184 ± 0.2095 ± (THC: 18.6/CBD: 0) 20.014 0.008 0.009 3

-   -   Tables 31a and b below show the amount of cannabinoid (in        milligrams) in the extract per gram pf plant material isolated        before and after winterization methods

TABLE 31a Amount of cannabinoid (in milligrams) in the extract per grampf plant material Before 1^(st) 1^(st) 2^(nd) winterizationwinterization winterization (mg/g) (mg/g) (mg/g) Strains Runs CBD Δ⁹-THCCBD Δ⁹-THC CBD Δ⁹-THC Variety 1 1 92.9 65.1 98.3 76.2 157.4 109.0 (THC:2 114.9 82.3 114.3 91.5 124.7 93.7 7.18/CBD: 3 115.5 85.7 113.7 93.8184.5 138.1 8.6) Variety 2 1 200.5 113.7 149.7 (THC: 0/CBD: 2 211.6109.5 147.3 9) 3 146.9 131.2 161.9 Variety 3 1 406.1 207.7 235.4 (THC: 2185.6 274.8 236.2 18.6/CBD: 0) 3 232.9 301.0 283.4

TABLE 31b Average cannabinoid (in milligrams) in the extract per gram pfplant material Before 1^(st) winterization 1^(st) winterization 2^(nd)winterization (mg/g) (mg/g) (mg/g) Strains Runs CBD Δ⁹-THC CBD Δ⁹-THCCBD Δ⁹-THC Variety 1 1 107.8 ± 12.9 77.7 ± 11.0 108.8 ± 9.1  87.2 ± 9.6155.5 ± 29.9 113.6 ± 22.6 (THC: 2 7.18/CBD: 3 8.6) Variety 2 1 186.3 ±34.6 118.1 ± 11.5 153.0 ± 7.8  (THC: 0/CBD: 2 9) 3 Variety 3 1 274.9 ±116.1 261.2 ± 48.1 251.7 ± 27.5 (THC: 2 18.6/CBD: 0) 3

Summary of Results:

Winterization of extracts was successful in removing waxes from theextract.

Experiment 11: Chemistry of Medicinal Cannabis Before and afterDecarboxylation

Methodology

Extraction:

Dried plant material (1 g) was weighed and transferred to a mortar andwas macerated using a pestle. The crused plant material was thentransferred into a 10 mL vessel and was subjected to supercritical fluidextraction (SFE), with supercritical CO₂ as solvent A and ethanol assolvent B. The photodiode array detector was set to monitor wavelengthsin the range of 200-600 nm and the back pressure regulator was set to 12MPa. The SFE conditions used were: flow rate=10 mL/min (CO₂ and slavepumps) and 1 mL/min (make-up pump); temperature=25° C.; gradient: 100%A−50% A (0.1-25 mins), 100% B (25-26 mins) and 100% A (26-30 mins). Oncethe method was completed, all fractions were combined and concentratedto dryness under reduced pressure (at 25° C.) to afford 0.28 g of agreen sticky resin. This was used for further work-up and analyses.

Activation:

Activation of phytocannabinoids was conducted by subjecting cannabisextract to heat using microwaves. A 5 mL-size microwave vial was chargedwith cannabis extract (27.72 mg) dissolved in ethanol (2 mL). The vialwas sealed and was subjected to heat for 10 min at 150° C. in a pressurevessel to afford a green sticky extract. This was concentrated todryness at 35° C. to obtain the activated cannabis extract as a resin(21.2 mg).

FIGS. 28A and 28B show the overview of select signals as seen in themass spectra before (A) and after (B) decarboxylation of cannabisextract, as well as their corresponding compound classes. Bolded signalsare compounds identified in the cannabinoid biosynthetic pathway.

Table 32.

Potential changes in chemical composition after decarboxylation ofstrain I cannabis extract. Left column indicates the potential compoundsthat were present in cannabis extract obtained through a supercriticalfluid extraction (SFE), but not in the decarboxylated resin. Thisextract was then subjected to heating conditions using microwavetechnology, and the right column shows the new chemicals that wereidentified, which were not present in the cannabis extract prior toemploying microwave technology described in this disclosure.

TABLE 32 New Compounds Found Upon Compounds Lost Upon ActivationActivation Roughanic acid (Fatty acid) Kaempferol (Flavonol) α-Linolenicacid (Fatty acid) Luteolin (Flavonol) Quercetin (Flavonoid)4,7-Dimethoxy-1,2,5-trihydroxyphenanthrene (Non-cannabinoid)4,5-Dihydroxy-2,3,6-trimethoxy-9,10-5-Methyl-4-pentyl-2,6,2-trihydroxybiphenyl dihydrophenanthrene(Non-cannabinoid) (Non-cannabinoid) Δ⁹-Tetrahydrocannabiorcolic acid(C₁- 5-Methyl-4-pentylbiphenyl-2,2,6-triol (Non- Cannabinoid Acid)cannabinoid) Cannabigerol (C₅-Neutral Cannabinoid) Cannabichromevarin(C₃-Neutral Cannabinoid) Cannabichromanon (Neutral Cannabinoid)Cannabicyclovarin (C₃-Neutral Cannabinoid) Cannabigerovarinic acid(C₅-Cannabinoid Acid) Cannabidivarin (C₃-Neutral Cannabinoid)6,7-cis/trans-Epoxycannabigerol (CannabinoidΔ⁷-cis-iso-Tetrahydrocannabivarin (C₃-Neutral Derivative) Cannabinoid)7-Hydroxycannabichromane (Cannabinoid Δ⁹-tetrahydrocannabivarin(C₃-Neutral Derivative) Cannabinoid) C₄-Tetrahydrocannabinolic acid(C₄-Cannabinoid Chrysoeriol (Flavone) Acid) Cannabitriol (NeutralCannabinoid) C₄-Cannabidiol (C₄-Neutral Cannabinoid) Cannabiripsol(Neutral Cannabinoid) C₄-Tetrahydrocannabinol (C₄-Neutral Cannabinoid)Cannabigerolic Acid (C₅-Cannabinoid Acid) Cannabifuran (NeutralCannabinoid) 7R-Cannabicoumaronic Acid (Cannabinoid Acid) Cannabinol(C₅-Neutral Cannabinoid) Tetrahydrocannabinolic acid-8-one (CannabinoidCannabinodiol (C₅-Neutral Cannabinoid) Acid Derivative)5-Acetoxy-6-geranyl-3-n-pentyl-1,4- 10-oxo-Δ^(6a)-tetrahydrocannabinol(Cannabinoid benzoquinone (Non-cannabinoid) Derivative)4-Acetoxycannabichromene (Cannabinoid 7R-Cannabicourmarone (NeutralCannabinoid) Derivative) Cannabielsoic Acid-α/β (Cannabinoid Acid)Cannabichromanone-D (Neutral Cannabinoid)6,7-trans/cis-Epoxycannabigerolic acid Cannabidiol Monoethylether(Cannabinoid (Cannabinoid Acid Derivative) Derivative)Δ⁹-Tetrahydrocannabinolic acid + C₂H₂O Cannabielsoic Acid-A/B(Cannabinoid Acid) (Cannabinoid Acid Derivative) Orientin (Flavone)Sesquicannabigerol (Cannabinoid Derivative)4-Terpenyl-Δ⁹-Tetrahydrocannabinolate (Cannabinoid Acid Ester)α-Terpenyl-Δ⁹-Tetrahydrocannabinolate (Cannabinoid Acid Ester)Bornyl/epi-bornyl-Δ⁹-Tetrahydrocannabinolate (Cannabinoid Acid Ester)α/β-Fenchyl Δ⁹-Tetrahydrocannabinolate (Cannabinoid Acid Ester)

Closed system, microwave extraction provided the simultaneous extractionand decarboxylation of the cannabinoids. In the native cannabis extract,63 compounds could be observed (FIG. 28A), and in the decarboxylatedcannabis extract, there could be up to 22 new compounds (Table 32). Upto 26 compounds from the resin were not present in the decarboxylatedcannabis resin, but were present in the cannabis resin prior todecarboxylation step.

Experiment 12: Solvent-Free Decarboxylated Cannabis Resin

General Procedure

To remove the solvent from the decarboxylated resin, the followingprocedures are used:

distillers or rotary evaporators are used to evaporate solvents employedin extraction and decarboxylation process, concentrate and obtainsolvent-free decarboxylated resin. During the evaporation of solvent, ahigher temperature than ambient temperature is used to facilitate fasterevaporation of the solvent. In addition, a vacuum may be used tofacilitate removal of solvent at lower pressure than the atmosphericpressure. In general, such processes are well established and known tothose skilled in the art.

The resulting decarboxylated cannabis resin may comprise less than 5%solvent, or the resin can be solvent-free.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Numerical data may be presented herein in a range format. It is to beunderstood that such a range format is used merely for convenience andbrevity and should be interpreted flexibly to include not only thenumerical values explicitly recited as the limits of the range, but alsoto include all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited. For example, a numerical range of about 1 to about 4.5 shouldbe interpreted to include not only the explicitly recited limits of 1 toabout 4.5, but also to include individual numerals such as 2, 3, 4, andsub-ranges such as 1 to 3, 2 to 4 etc. The same principle applies toranges reciting only one numerical value, such as “less than about 4.5,”which should be interpreted to include all of the above-recited valuesand ranges. Further, such an interpretation should apply regardless ofthe breadth of the range or the characteristic being described.

While the foregoing disclosure has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art, from a reading of the disclosure that variouschanges in form and detail can be made without departing from the truescope of the disclosure in the appended claims.

All publications, patents, and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A decarboxylated cannabis resin, wherein the resin comprisesdecarboxylated cannabinoids from a Cannabis spp. plant, such thatΔ9-tetrahydrocannabidiolic acid (Δ9-THCA) and cannabidiolic acid (CBDA)cannabinoids in the plant are each independently 90% to 100%decarboxylated to yield Δ9-tetrahydrocannabinol (THC) and cannabidiol(CBD) respectively in the resin.
 2. The decarboxylated cannabis resin ofclaim 1, wherein the resin further comprises cannabinol (CBN).
 3. Thedecarboxylated cannabis resin of claim 1, where the resin issubstantially free of solvent.
 4. A pharmaceutical product comprising(i) the decarboxylated cannabis resin of claim 1 and (ii) a suitablepharmaceutically acceptable carrier or excipient.
 5. A natural healthproduct comprising the decarboxylated cannabis resin of claim
 1. 6. Amethod of extracting and decarboxylating cannabinoids from cannabis, themethod comprising: (i) extracting cannabinoids by contacting thecannabis with a solvent thereby forming a cannabis extract; and (ii)decarboxylating the cannabinoids in the cannabis extract by subjectingthe cannabis extract to microwaves at temperature of about 100-200° C.in a sealed container for a time period sufficient to form thecorresponding decarboxylated cannabinoids in the cannabis extract. 7.The method of claim 6, wherein before the step (i) of extracting, thecannabis is broken down to produce cannabis of a size and form suitablefor extraction.
 8. The method of claim 6, wherein the extracting anddecarboxylating steps occur concurrently.
 9. The method of claim 6,wherein the solvent is selected from the group consisting of: 80-100%ethanol, ethylene glycol, isopropanol, and a combination of any of theabove.
 10. The method of claim 6, wherein the cannabis is completelysubmerged in the solvent in the sealed container.
 11. The method ofclaim 6, wherein the time period is about 15-75 minutes and wherein thetemperature is between about 130° C. to about 180° C.
 12. The method ofclaim 6, wherein the microwaves have a frequency of about 2.45 GHz. 13.The method of claim 6, wherein the decarboxylating step (ii) occursunder a pressure of about 2-22 bar.
 14. The method of claim 6,comprising the step of (iii) removing the solvent from the cannabisextract, thereby producing a cannabis resin.
 15. The method of claim 6,wherein the extracted and decarboxylated cannabinoids are recovered inthe form of isolated compounds.
 16. The method of claim 14, wherein thecannabis resin is subjected to winterization for the removal of waxes.17. The method of claim 1, wherein the cannabis is dried cannabis. 18.(canceled)
 19. The method of claim 6, wherein the cannabis is a plantpart selected from the group consisting of a trichome, a cannabis femaleinflorescence, a flower bract, a cannabis stalk, a cannabis leaf, andcombinations thereof.
 20. (canceled)
 21. The method of claim 6, whereinthe cannabis remains in contact with the solvent during thedecarboxylating step.